Identification of Human Genetic Variants for High Risk of Severe Influenza Disease
Adrianus Boon, Ph.D.
Influenza virus kills nearly 500,000 individuals each year and this number can increase dramatically during a pandemic with a novel strain of influenza virus. Infections with influenza virus can be prevented by antiviral drugs like oseltamivir or vaccinations. However, antiviral drugs are not prescribed prophylactically and vaccination is not monitored for efficacy providing ample opportunity for influenza to infect and cause disease. If we can identify individuals that are at increased risk of severe influenza virus we can tailor our care to provide drugs prophylactically to improve health and longevity. The goal of our research is to define genetic biomarkers that predict susceptibility to influenza disease. The interferon pathway is very important for restricting influenza virus replication and therefore disease. Individuals with unique or rare polymorphisms in essential genes in this pathway are more susceptible to influenza virus and more likely to require hospital care or succumb to infection. Because of the importance of this pathway we want to study polymorphisms in two interferon pathway genes that have previously been shown to effect viral diseases and antiviral immunity. These polymorphisms are relatively common in the population with about 5-10% of all people carrying two copies of the minor allele. To test if these genetic differences between individuals predispose to more severe disease we will use cultured primary human airway epithelial cells as a model to look at the role of these polymorphisms on disease parameters. Airway epithelial cells are the primary target cell of influenza virus and we know that increased virus replication and attenuated antiviral immunity correlate with severe disease. We will determine virus replication and antiviral gene-expression in cells obtained from individuals with different IFN genotypes. Analysis of the data between cells containing the two alleles will reveal if this genetic marker can be used to identify individuals that are genetically susceptible to influenza virus. The identification of prognosticators of influenza disease and survival is of special interest to the Foundation and leads to fewer influenza associated deaths and hospitalizations.
Personalized and precision medicine is been made possible by the advent of genomic medicine. Cancer treatment in particular has made significant progress towards the improvement of patient care, increased survival and reduced costs, but progress has lagged in other diseases. Nonetheless, we propose that genomic medicine can also impact the treatment or prevention of other life-threatening diseases such as influenza. Influenza virus kills nearly 500,000 individuals each year and this number can increase several-fold during an influenza pandemic Epidemiologic studies have identified rare genomic polymorphisms in the antiviral interferon pathway that are linked to severe influenza infections and hospitalizations. Interferons are secreted proteins that induce cell-intrinsic antiviral immunity in virus-infected and uninfected neighboring cells. The effect of common polymorphisms in genes in this pathway on influenza susceptibility has not been evaluated. To identify genetic biomarkers that can be used to identify susceptible individuals we will culture human airway epithelial cells harboring selected genotypes in two genes involved in the cell-intrinsic antiviral immune pathway. Virus replication and antiviral gene expression will be quantified and compared between cells containing the different genotypes. Differences in virus replication or antiviral immunity will strongly suggest that these polymorphisms can be used as biomarkers to identify individuals at increased risk of developing severe influenza. These susceptible individuals should receive antiviral drugs during influenza outbreaks.
Genotyping of cohort: With our collaborators (Dr. Steven Brody at Washington University and Dr. Matthias Salathe at the University of Miami) we cultured and expanded human primary airway epithelial cells from 23 different donors. We also developed genotyping protocols for polymorphisms in the IRF7 (rs12805435) and IFNL3 (rs12979860) host genes. Among the 23 samples, we identified 10 donors that were homozygous for the major allele (T) of IRF7, 8 donors that were homozygous for the minor allele (C), and five that were heterozygous. Fourteen samples were also genotyped for IFNL3 and we identified 6 samples that were homozygous for the major allele (C), 4 that were homozygous for the minor allele (T) and four that were heterozygous.
Analysis of influenza A virus growth in primary airway cultures: Cells from fourteen different donors were selected based on the genotype for IRF7 and were homozygous for the major or minor allele. Confluent monolayers were inoculated with a low dose of A/California/04/2009 H1N1 virus and supernatant from the infected cells was collected at 24, 48 and 72 hours post-infection. The viral titer in the supernatant of these cells was quantified by virus titration assays. At 24 hours post infection, we observed a more than 1000-fold difference in virus titer between cells derived from different donors. This difference was maintained at 48 and 72 hours post infection. These findings suggest a role for host genetic variation on influenza virus replication in primary airway epithelial cells.
Association between polymorphisms in IRF7 and IL28b and influenza virus replication: The average virus titer was compared between the different IRF7 and IFNL3 genotypes. We found no difference in virus titer from cells that were homozygous for the major or minor allele of IRF7. Similarly, we also observed no difference in virus titer from cells that were homozygous for the major or minor allele of IFNL3. Interestingly, the four samples that were heterozygous for IFNL3 produced the least amount of influenza virus at 24 hours post infection. These data suggest that polymorphisms in IRF7 do not impact virus replication in primary airway epithelial cells in vitro. The impact of genetic variation in IFNL3 is unclear and warrants further investigation.