br Materials and Methods br Results br
Materials and Methods
Discussion Investigations by our group and others have shown differential expression of host genes at the RNA level in peripheral blood in response to ARV infections (Ramilo et al., 2007; Zaas et al., 2009; Woods et al., 2013; Zaas et al., 2013), with heavy representation of genes in the IFN-signaling canonical pathway and innate immune response signaling. Analysis of differentially expressed NPL proteins in infected individuals demonstrates involvement in several biological pathways critical to mounting a host defense to virus infection, including innate immune responses, acute inflammatory responses, and defense response pathways. The inclusion of three members of the complement system (CFAB, A1AT, and IC1) is particularly consistent with an innate immune response, as the complement system enhances the ability of mecamylamine and phagocytic cells to clear pathogens from the infected site (Ricklin et al., 2010). Despite the apparently related pathways involved, there does not appear to be extensive overlap between the differentially expressed nasal proteins identified in our H3N2 #1 challenge study, and the previous peripheral blood RNA signatures of ARV infection characterized in the same challenge cohort (Zaas et al., 2009). One gene product that is increased at both the NPL peptide and peripheral blood RNA level upon influenza infection (Cameron et al., 2008; Cameron et al., 2007; Zaas et al., 2009) is IC1 (SERPING1 gene). IC1 (also called C1-inhibitor) is a peptidase inhibitor belonging to the serpin superfamily and has an important role in innate immunity through modulation of the classical pathway of complement activation (Gaboriaud et al., 2004). As the complement system has the potential to be damaging to host tissues, complement control proteins must tightly regulate activation. IC1 binds to complement protein C1 to inhibit activation of the classical complement pathway, and thus its discovery fits well with our understanding of the biology of these diseases. Notably absent from this NPL protein analysis are proinflammatory cytokine and chemokine gene products which have previously been shown to be strong contributors to the host response both in peripheral blood and near the site of ARV infection (Kimura et al., 2013). Oshansky and colleagues assayed nasal lavage samples from a cohort of healthy and naturally-infected influenza patients using a multiplex cytokine and chemokine assay panel, reporting correlation of inflammatory cytokines MCP-3 and IFN-α2 with disease progression (Oshansky et al., 2014). An aptamer-based detection method was subsequently used to screen the same cohort and generate quantitative measures of over 1000 protein analytes from nasal lavage, showing differential expression of 162 proteins including cytokines associated with immune response to infection (Marion et al., 2016). We did not identify inflammatory cytokines to be differentially expressed in our pooled NPL analysis. It is possible that cytokine proteins in NPL samples are expressed at levels below the detection limits of LC/MS-based methods, and that coupling targeted methods capable of detecting and quantifying cytokines directly may provide enhanced datasets for biomarker discovery. Categorizing infection based upon host response represents an emerging strategy with great potential for complementing current pathogen-based diagnostics, as well as providing additional insights into the pathobiology of infection. The results presented in this study provide evidence that a protein-based host response to ARV infection can be detected in the nasopharyngeal space, and that this response involves perturbation of pathways involved in acute inflammation and innate immune response. Further, this work demonstrates that targeted assays measuring peptides involved in this response allow classification of ARV infection with a high degree of accuracy. Validation of these findings across independent experimentally infected Influenza A and human rhinovirus cohorts suggests a robust and generalized response to viral infection. With further development as a clinical diagnostic, this signature may have utility in rapid screening for emerging infections, avoidance of inappropriate antimicrobial therapy, and more rapid implementation of appropriate therapeutic and public health strategies. Nonetheless, as with other validated biomarkers, additional validation in community-based cohorts will be important to demonstrate the potential utility of such an assay in its clinical applications. Furthermore, testing this approach across a larger series of upper respiratory viruses will be important to understand its full potential utility and limitations. An assay that combines host protein biomarkers with nasal viral antigen detection may be quite valuable in clinical care to optimize therapeutic decision making. And whilst a positive result from such an assay may avoid the use of inappropriate microbial therapy, it will likely require vigilance on the part of the clinician to exclude bacterial co-infection when clinically indicated.