key: cord-1041974-qi5dnjlu authors: Liou, T. G.; Adler, F. R.; Cahill, B. C.; Cox, D. R.; Cox, J. E.; Grant, G. J.; Hanson, K. E.; Hartsell, S. C.; Hatton, N. D.; Helms, M. N.; Jensen, J. L.; Kartsonaki, C.; Li, Y.; Leung, D. T.; Marvin, J. E.; Middleton, E. A.; Osburn-Staker, S. M.; Packer, K. A.; Shakir, S. M.; Sturrock, A. B.; Tardiff, K. D.; Warren, K. J.; Waddoups, L. J.; Weaver, L. J.; Zimmerman, E.; Paine, R. title: SARS-CoV-2 Innate Effector Associations and Viral Load in Early Nasopharyngeal Infection date: 2020-11-04 journal: medRxiv : the preprint server for health sciences DOI: 10.1101/2020.10.30.20223545 sha: 6438b5ee5170ecbb985faac0fa3dbf8de27f7501 doc_id: 1041974 cord_uid: qi5dnjlu To examine innate immune responses in early SARS-CoV-2 infection that may change clinical outcomes, we compared nasopharyngeal swab data from 20 virus-positive and 20 virus-negative individuals. Multiple innate immune-related and ACE-2 transcripts increased with infection and were strongly associated with increasing viral load. We found widespread discrepancies between transcription and translation. Interferon proteins were unchanged or decreased in infected samples suggesting virally-induced shut-off of host anti-viral protein responses. However, IP-10 and several interferon-stimulated gene proteins increased with viral load. Older age was associated with modifications of some effects. Our findings may characterize the disrupted immune landscape of early disease. Part of the challenge to developing a treatment or prevention response is to elucidate the range of innate immune responses to SARS-CoV-2 infection that may precede the cytokine storm seen in severe disease. 24, 26 Evaluations in ARDS, especially due to severe respiratory viral infections such as Influenza A, 23, 27, 28 Middle Eastern Respiratory Syndrome, [29] [30] [31] SARS-CoV 32-37 and other highly pathogenic coronaviruses such as Human CoV-(HCoV)-Erasmus Medical Center (HcoV-EMC) [38] [39] [40] highlight the broad collection of biomarkers that may potentially be useful in SARS-CoV-2 infection, especially prior to fulminant disease, uncontrolled systemic inflammation and ARDS. Coronaviruses that cause severe human disease are remarkable for their ability to evade innate immune defenses and to promote dysfunctional responses that appear before cytokine storm. 41, 42 For example, IFN responses are critically important for anti-viral defense, 43 yet there is no detectable native human IFN response to SARS-CoV. 38 No fewer than 11 of 28 known SARS-CoV proteins interfere with signaling cascades that produce IFN proteins after endosomal or cytoplasmic viral detection. 44 In vitro studies of various human cell types show that SARS-CoV efficiently suppresses transcription of IFN genes but selectively allows expression and translation of other genes. [45] [46] [47] Bronchoalveolar lavage studies of epithelial cells obtained from severely ill patients following SARS-CoV-2 infection suggest a pathophysiology more consistent with HCoV-EMC or MERS-CoV than SARS-CoV. 21 However both SARS-CoV and SARS-CoV-2 stimulate inflammatory signals via nuclear factor κB (NFκB) 44 that recruit polymorphonuclear neutrophils and other immune effector cells to the lung, releasing proteases that may dramatically further increase viral cell entry. [48] [49] [50] IFN-α and IFN-β treatments that bypass some evasion strategies 21 have been proposed to counter both SARS-CoV viruses. [51] [52] [53] However, we lack efficacy and safety trials free of observer bias, 54 and no published human data exists for IFN-λ therapy. To supplement the growing information on responses early in infection, we undertook an observational study of deidentified nasopharyngeal swab samples from patients presenting at drive-through testing centers for evaluation of symptoms potentially due to SARS-CoV-2 infection. We selected proteins involved in different for understanding viral entry, intracellular detection of viral invasion, production of pro-inflammatory signals, systemic inflammatory agents and multiple IFN and IFN-stimulated gene (ISG) responses relative to viral loads to better understand the immune landscape of patients with early disease. We evaluated 40 samples from individuals, evenly divided into 20 positive and 20 negative detection results for SARS-CoV-2. Samples were deidentified but annotated by age (median 46.5 years, range 11-90) and sex (17 females, 42.5%). Older patients were more likely to be male and negative for SARS-CoV-2 detection (Supplementary Figure 1 and Supplementary Table 1a and 1b). This limited demographic information suggested that further evaluation of statistical relationships in our sample set required testing adjustments for age, sex or both to avoid confounding. Samples included in our study were randomly selected from those collected from April-June of 2020 from patients who may have come from nine states within the Mountain West of the United States. During this period, positive results were reported for about 9-10% of tested patients. Among the positive test patients, about 10% eventually required hospitalization for COVID-19 with less than 50% of those hospitalized suffering respiratory failure, ARDS or succumbing to severe disease. While we know this context for our samples, the specific outcomes for individual patients in our study are unknown. Using RT-PCR results, we estimated fold-change in mRNA expression of SARS-CoV-2 small envelope protein E1 (Odds Ratio [OR] = 10.8 × 10 6 , 95% Confidence Interval [CI] = 8.37 × 10 5 -1.40 × 10 8 , p < 0.001) and nucleocapsid protein N1 (OR = 5.1 × 10 7 , CI = 4.5 × 10 6 -5.9 × 10 8 , p < 0.001) relative to expression in patients without infection. We selected primers 55 for E1 originally from Charité, Germany 4 and N1 from the US CDC. 56 6 Both E1 and N1 mRNA fold changes gave virtually total discrimination between patients with and without infection diagnosed by clinical testing for SARS-CoV-2 infection using qualitative RT-PCR. We measured two human protein transcripts important for understanding SARS-CoV-2 cell entry, angiotensin converting enzyme-2 (ACE-2), which is essential for entry of SARS-CoV-2 and SARS-CoV, 48, 57 and transmembrane protease, serine-2 (TMPRSS-2) which enhances cell entry up to a thousand-fold. 48,49 ACE-2 mRNA was increased three-fold in patients with infection, and the fold-change results were strongly associated with viral load. TMPRSS-2 mRNA expression, however, was not associated with infection nor viral load ( Figure 1 ). We examined transcription signals for two genes in the signaling pathway downstream of viral detection important for IFN responses, TNF-associated factor-binding kinase-1 associated with inhibitor of NFκB (TBK-1) and Stimulator of IFN genes-(STING)-1 for six patients with positive detection of SARS-CoV-2 and six patients with negative detection. The mRNA expressions of TBK-1 and STING-1 were not associated with infection (Supplementary Figure 2) . both viral protein E1 mRNA (OR = 0.945, CI = 0.906-0.986, p = 0.025) and N1 mRNA (OR = 0.947, CI = 0.907-0.989, p = 0.034, Figure 2e ). We found NFκB-1 and NFκB-2 mRNA transcripts were not significantly changed compared to uninfected status (Table 1a) . Other downstream immune effectors, granulocytemacrophage colony stimulating factor (GM-CSF), IL-6 and IL-10 mRNA were not increased (Table 1a) . Protein measurements using bead-based multiplex immunoassays (BioLegend) for systemic inflammatory markers matching many of the mRNAs measured (plus IL-1b and IL-12p70) revealed no significant changes with infection and no significant associations with viral load with one exception. IP-10 protein was increased nearly four fold above measured control values (Table 1b) , and the log of concentration was strongly associated with viral load (OR = 1.09 per unit of log unit of viral N1 mRNA fold-change, CI = 1.06-1.12, p < 0.001). We found five to six-fold increases in expression of both IFN-λ1 and IFN-λ2 mRNA among patients with detection of SARS-CoV-2 (n=20) compared to patients without detection of virus (n=20) (Table 2a, Figure 3a and 3c). There were no other significant increases in IFN mRNA. The increases in IFN-λ1 and IFN-λ2 mRNA production were strongly associated with viral load (Figure 3e and 3f). Despite increased mRNA expression for some of the IFNs, protein measurements showed reductions in IFN-α2, IFN-γ and IFN-λ2,3 in patients with viral infection that averaged 66%, 49% and 40%, respectively, relative to control patients (Table 2c) . significantly increased (Table 2d) . Focused proteomic examination of proteins extracted from samples using data independent acquisition (DIA) mass spectrometry detected an association between positive clinical testing for SARS- CoV-2 and IFIT-1, IFIT-3 and Tetherin proteins with a borderline finding for MX-1 protein (Table 2e ). Proteomic examination using data dependent acquisition (DDA) mass spectrometry, which has less sensitivity but better specificity and precision, detected only a large increase in IFIT-3 protein that was associated with clinical infection detection and increasing viral N1 mRNA (Figure 4 ). Transcript and proteome results are based on the same six positive and six negative samples for which we had sufficient mRNA remaining after other studies. The increase in IP-10 mRNA with infection was lower in older individuals ( Figure 2i ). All other things being equal, 10 additional years in age were associated with an approximately overall 70% reduction in IP-10 mRNA compared to controls while 25 additional years in age were associated with an overall 90% reduction, producing a remarkable counter effect to infection itself. We found a borderline significant effect for IFN-λ2,3. Patients with older ages have slightly lower IFN-λ2,3 per additional year of age with SARS-CoV-2 infection. This small per year effect was associated with a 10% lower IFN-λ2,3 on average for every additional 10 years of age and about 23% lower IFN-λ2,3 for an additional 25 years of age in addition to the approximately 40% reduction associated with SARS-CoV-2 infection (Table 2c) . correlations within IFN and inflammatory proteins (Supplementary Table 2c ). Correlations within IFN types, for example, among IFN-λ sub types were exceptionally strong which corresponds to the biology of the IFNs. We performed sensitivity testing for all significant associations between IFN, inflammatory and ISG measurements with viral load (N1 protein mRNA) by re-examining the relationships after exclusion of patients without infection by SARS-CoV-2. In every case, we found similar relationships between each biomarker and viral load, increasing the confidence in our findings. Because of the high degree of correlation between some biomarkers (Supplementary Table 2 ), for example between IFN-β1 and IFNα2 transcripts (Spearman correlation coefficient = 1.00, p < 0.001), we examined the effect of adding a second biomarker as an adjustment to the relationship between each significant biomarker (p < 0.05) with the clinical diagnosis of infection (Tables 1a, 1b, 2a, 2c and 2d) or with viral load (Tables 2b and individually reported results for ACE2 mRNA, IP-10 mRNA and IP-10 protein). In every case, we found similar results for the association between the biomarkers reported and infection status or viral load. A number of the biomarker measurements tested as adjustment variables appeared to have independent significant effects suggesting that significant and independent multivariable associations exist, however, our study is too small to report those results with confidence. Our results show large increases in transcription of multiple genes involved in innate immune and inflammatory responses soon after SARS-CoV-2 infection and the development of viral-like symptoms (Tables 1 and 2 and Figures 2 and 3) . However, there was a broad-based discrepancy in translation response relative to increased transcription signals similar to the host shut off patterns seen with multiple viruses, including human CoVs such as SARS-CoV that have been reported by many and reviewed by others, [70] [71] [72] [73] [74] and that is just beginning to be 10 All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted November 4, 2020. ; https://doi.org/10.1101/2020.10.30.20223545 doi: medRxiv preprint described in SARS-CoV-2. 75, 76 An alternative possibility is that proteins are rapidly degraded after translation, however, either possibility is detrimental to a fully functional innate immune response. Among the IFNs that we evaluated, several had large increases in transcription that were also strongly associated with viral load (Table 2a and 2b), but protein production was either unchanged or decreased when comparing samples from symptomatic infected patients to uninfected controls (Table 2c) . For pro-inflammatory cytokines, there were similar large increases in transcription (Table 1a ) but no change in measured protein production except for IP-10 alone (Table 1b ). Considering that our samples were collected soon after initial symptoms from ambulatory patients, the protein production result may indicate that IP-10 is among the first inflammatory proteins to increase early in infection. The discrepancies between transcription and translation did not fully extend to the ISGs. We selected to evaluate these molecules because of their importance for anti-viral defense. [60] [61] [62] [63] [64] [65] [66] [67] [68] Observed enormous increases in transcription (Table 2d) were accompanied by several large but uncorrelated (Supplemental Table 2 ) increases in protein production (Table 2e ). Three antiviral ISGs had increased transcription and translation: IFIT-3 most strongly ( Figure 4 ), IFIT-1 and Tetherin, and there was an additional borderline finding for MX-1 (Table 2e ). In SARS, suppression of anti-viral proteins occurred late in clinical disease, 77 however, our results suggest that with SARS-CoV-2, it occurs near the beginning of symptoms. Host translation suppression in SARS is associated with spike protein and non-structural protein 1 (NSP1) interactions with eukaryotic initiation factor-(eIF)-3 which is required for protein translation. 72 Two recent publications investigating mechanisms involving Nsp1 for SARS-CoV-2 showed similar interference with eIF-3. 75, 76 Our results add to the in vitro work by demonstrating supportive evidence from early in the clinical course of human infection. Because viruses depend on host mechanisms for translation of viral proteins that are required for assembly of new infectious particles, our observation of continuing transcription and suppressed translation of human proteins may help explain persistent RT-PCR detection of viral RNA but marked decreases in infectious viral particle production soon after the appearance of symptoms. These tentative hypotheses await further development and testing. All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted November 4, 2020. ; https://doi.org/10.1101/2020.10.30.20223545 doi: medRxiv preprint ACE-2 mRNA was increased among patients with SARS-CoV-2 infection. The finding indicates at least two possible causal relationships. SARS-CoV-2 may selectively infect people with existing high levels of ACE-2 transcription, or infection itself may increase transcription of ACE-2 above normal. In either case, increased transcription leading to increased protein expression of ACE-2 likely would increase viral entry and thus help amplify viral replication. We measured transcripts for three intracellular proteins important in pathways leading to IFN production and initiation of NFκB-related inflammation. Transcription of TBK-1 and STING-1 were unchanged while there was a decrease in TRAF-1 mRNA. TRAF-1 is involved in several distinct inflammation-related pathways, but a reduction is most likely associated with increased NFκB activity and subsequently increased systemic inflammation. 58 The other two proteins, TBK-1 and STING-1, are important for transmitting detection of viral invasion to processes that produce anti-viral IFNs. 41 There was no increase in transcription of STING-1 and TBK-1, however, IFN-λ1 and IFN-λ2 transcripts were markedly elevated (Table 2a and Figure 3 ). The increases in these transcripts were closely associated with viral load. These findings suggest that detection of viral invasion is successful in generating a signal to increase both systemic inflammation and IFN production. Massive and strongly significant increases in IFN-λ1 and IFN-λ2 mRNA (Table 2a ) may indicate the critical importance of Type III IFNs in SARS-CoV-2 78-80 even if protein production was decoupled from high levels of gene transcription by the time our samples were collected. IP-10 was the sole inflammatory cytokine detected with higher protein concentrations in our samples from infected patients (Table 1b) . IP-10 promotes inflammation in Human Immunodeficiency Virus, 81 H5N1 Influenza A, 82, 28 Middle-East Respiratory Syndrome virus 83 and SARS-CoV 33 infections, thus its prominence early in SARS-CoV-2 infection, while unsurprising, may be important for understanding evolution of disease from initial mildly symptomatic to severe and sometimes fatal. Nevertheless, we found a moderately strong inverse relationship with age such that 10 or 25 additional years of age seemed to be associated with dampening of increases in IP-10 (see Results). This inverse association is at odds with the clinical observation of worsening disease severity associated with older ages and generates questions about the nature of previously observed 12 All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted November 4, 2020. ; https://doi.org/10.1101/2020.10.30.20223545 doi: medRxiv preprint detrimental effects of IP-10 14-17 on morbidity and mortality with SARS-CoV-2 infection. Older individuals with COVID-19, for example, may be more sensitive to suppression of anti-viral defenses while younger individuals are more sensitive to excessive inflammation. The observations and questions show that abnormal transcript and protein responses to infection cannot be fully interpreted without clinical context drawn from evaluation of a larger study population. In contrast to limited but interesting results with adjustments for age, adjustments for sex were uninformative. The lack of significant findings may be due to survivor biases. Ill and severely ill patients are less likely to be female, 15 but the susceptibility to infection associated with sex is unknown. Among patients who develop symptoms, innate immune responses may be similar regardless of sex. Our study is limited by its cross-sectional design, small size and the nature of the nasopharyngeal swab samples. Due to the urgency of need, we obtained deidentified samples quickly in exchange for giving up detailed clinical annotation. We do not yet have sufficient information to interpret observed abnormalities in IFN and systemic inflammation to seek out associations with clinical outcomes such as respiratory failure or death. However, because a random sample of the population visiting our drive-through diagnosis centers will contain predominantly survivors of infection who never require hospitalization, the measurements we report should roughly represent patients who generally suffer non-severe disease. The small size of the study limits our ability to generalize our interpretations and conclusions. Sensitivity analyses, however, increase our confidence in the stability of our findings and suggest that there are additional multivariable associations between biomarkers, infection status and viral load that may be explored, further strengthening the impression that additional study of more individuals is needed to better understand the extent of innate immune disruptions due to SARS-CoV-2 infection. Nasopharyngeal swab samples necessarily retrieve a variety of cell types and may retrieve secreted substances that originate elsewhere than the upper airway. Because samples were frozen prior to evaluation, characterization of cell types by cell counting or flow cytometry was not possible. Prospective human study with immediate processing to allow better assessments of cell types present is possible, but full characterization 13 All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted November 4, 2020. ; https://doi.org/10.1101/2020.10.30.20223545 doi: medRxiv preprint of secreted molecules will require carefully designed cell culture models to prevent inclusion of molecules produced elsewhere and transported to the nasopharynx. Despite the limitations, our study provides information highlighting several areas of IFN and inflammatory biology that deserve future investigation. Although our study identifies strong IFN and systemic inflammatory signal transcription responses to infection, only a larger prospective study incorporating careful annotation of patient characteristics, analysis of serial samples with disease progression and reporting of outcomes can fully assess the clinical implications of these initial findings. Our results overall, even with a small study size, emphasize that there are remarkable disruptions early in disease in the immune landscape. Further study is likely to be both fruitful and illuminating. Our project was reviewed at the University of Utah by both the Institutional Review Board and the Biosafety Committee. An exemption from informed consent was allowed because patient samples were de-identified. All samples were handled in a biosafety level (BSL) 2 capable hood (ThermoFisher Scientific, Waltham, MA, USA) using BSL 3 procedures until virus inactivation and were handled with BSL 2 procedures thereafter. Randomly selected and completely deidentified, residual nasopharyngeal swab samples from patients presenting for diagnosis of symptoms consistent with COVID-19 during the period of late April through early June of 2020 were enrolled in the study. Clinical testing involved use of a portion of each sample to test with automated, FDA Emergency Use Authorized RT-PCR or transcription-mediated amplification tests for qualitative presence of SARS-CoV-2 RNA. We received sample remainders annotated with age, sex and qualitative nucleic acid amplification-detection results after being frozen at -80 °C for approximately one month. 14 All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted November 4, 2020. We carefully aspirated the supernatant for Bead Based Multiplex Immunoassays. Pellets from centrifugation were extracted using All-Prep Micro kits (Qiagen) in accordance with the manufacturer's instructions, producing additional RNA suitable for RT-PCR and a final protein-containing pellet for Mass Spectrometry. For most mRNAs, we had sufficient sample to study all 40 patients; for selected mRNAs, we were able to study 6 samples with and 6 samples without SARS-CoV-2 detection. All specific mRNA measurements were based on RT-PCR employing RNA from a single extraction method to avoid technical sources of noise. An equal volume of RNA was taken for first strand cDNA reverse transcription (ABI High Capacity cDNA Reverse Transcription Kit) and specific amplification in a StepOnePlus (ABI, ThermoFisher Scientific). Gene specific primers were designed using the Roche Applied Science Universal Probe Library Assay Design Center. All amplifications were performed using a 2-step amplification protocol with ABI PowerUp SYBR Green Master Mix as follows: 1 cycle at 50° C for 2 minutes to activate UDG, 1 cycle at 95° C for 2 minutes to release the DNA polymerase then 40-50 cycles with a 3 second denaturing at 95° C followed by 30 second annealing and denaturing at 60° C. A melt curve (dissociation) was performed for every primer to ensure the above amplification conditions resulted in the amplification of a single peak. All of the designed primers gave a single peak upon dissociation 15 All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted November 4, 2020. ; https://doi.org/10.1101/2020.10.30.20223545 doi: medRxiv preprint after amplification suggesting no non-specific binding to other genes. Amplification of genomic DNA was prevented by using primers that spanned an intron. The IFN-α2 gene and IFITM-1 and IFITM-3 ISGs do not have introns. The primers did, however, give a single peak upon dissociation. All other primers including IFN-λ spanned an intron. Cytokine analyses of patient samples were performed using a commercially available enzyme-linked 16 All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted November 4, 2020. ; https://doi.org/10.1101/2020.10.30.20223545 doi: medRxiv preprint Preparation of proteins prior to mass spectrometry. Proteins were reduced with 5 mM dithiothreitol (DTT) at 60° C for 45 minutes, followed by alkylation with 10 mM iodoacetamide (IAA) at room temperature for 30 minutes in the dark. Excess IAA was neutralized by addition of 5 mM DTT. A trypsin/LysC mixture (Promega; Madison, WI) was added to the proteins in a 1:100 ratio and the proteins were digested overnight at 38° C. The digestion was quenched by acidification of the solution with the addition of 1% formic acid to a pH of 2-3. Initially, the pelleted proteins from the COVID-19 patients would not completely dissolve in the 50 mM ammonium bicarbonate. However, after the trypsin/LysC digestion all of the samples were completely dissolved in solution. The final concentration of the peptides was determined using a peptide colorimetric assay and the use of a Nanodrop One (ThermoFisher Scientific) spectrophotometer. Peptides (1 μg on column) were loaded using a Dionex UltiMate 3000 RSLCnano system (ThermoFisher Scientific) onto a PharmaFluidics μPAC micro-chip based trapping column and separated using a 50 cm equivalent PharmaFluidics μPAC micro-chip based column (PharmaFluidics, Ghent, Belgium). Chromatography was performed using ultrapure water with 0.1% formic acid (solvent A) and acetonitrile containing 0.1% formic acid (solvent B). Elution was carried out with an initial mobile phase concentration of 5% for 4 minutes followed by a ramp to 45% over 76 minutes then a second ramp to 95% B in 5 minutes. This was held for 10 minutes followed by ramping down to 5% B over two minutes and reequilibration for 10 minutes. Flow rate was 0.5 mL/min. A QExactive HF (ThermoFisher Scientific) coupled to a Flex nano spray source was employed with the following settings for MS1; resolution 60, AGC target 3e6, maximum IT 100 ms, scan range 375-1650 m/z. MS2 settings were; resolution 15,000, AGC target 2e5, maximum IT 25 ms, isolation window 1.4 m/z. Top 15 DDA analysis was performed with NCE set to 27. DIA nanoLC-MS/MS. Staggered window DIA analysis was carried out using the methods described by Pino et. al. 85 A peptide centric gas phase retention time library was generated by pooling equal amounts of each sample and analyzing this using six narrow window DIA experiments with the following settings for MS1: resolution 60,000, AGC target 1e6, maximum IT 55, with 6 separate analyses in the following mass ranges 395-17 All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted November 4, 2020. DIA data processing. Thermo .RAW files were demultiplexed and converted to mzML files using MSConvert. 86 The Walnut functionality of EnclopeDIA 87 was employed for peptide centric library creation. Peptides were identified using the same variables as DDA described above. Quantitation was performed using Skyline. 88 For all mRNA, we calculated fold-change for each sample (FCsample) after measuring the fractional number of polymerase chain reaction doubling cycles required so that SYBR Green fluorescence exceeded the threshold for detection (C T ). We used the following formula: where ΔC T sample was the number of doubling cycles to detect each mRNA minus the number of doubling cycles to detect mRNA from the Pol2A reference gene for each sample, and the ΔC T median was the median ΔC T sample for samples without detection of SARS-CoV-2. The incorporation of C T for Pol2A mRNA in the 18 All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted November 4, 2020. ; https://doi.org/10.1101/2020.10.30.20223545 doi: medRxiv preprint calculation indexes the measurement so that samples with different efficiencies of recovery of mRNA containing cells between testing individuals are standardized. For IFIT-3 fold change values from DDA mass spectrometry, FCsample was calculated: where AUC IFIT3 is the area under the curve (AUC) for peptides identified as part of IFIT-3, and median(AUC virus negative IFIT3 ) is the median AUC for IFIT-3 from samples without detection of SARS-CoV-2. The other prespecified ISG proteins were undetectable by DDA mass spectrometry, and thus no fold-change calculation was possible. We calculated summary statistics. We examined associations between different biomarker measurements by calculating Spearman's rank correlation statistic to better understand potential dependencies. We used log transformations of all mRNA and protein measurements in our statistical calculations because of the log-normal nature of our results. Others using the methods that we employed, however, often report results using either natural or base 2 logs. Because the bulk of our results are commonly reported using natural log values, we standardized on those for reporting. The effect is to slightly change results normally reported using base 2 logs by a proportion equal to natural log of 2 (or 0.698). This usage has no effect on interpretations of results. Using SARS-CoV-2 infection status as the independent variable, we performed linear regression with natural log(FC) of each pre-specified mRNA or natural log(FC IFIT3 ) as the dependent variable because we seek to understand the biological effects of infection. Each univariable model was adjusted with age and sex with backward selection to understand the impacts on model fits. We performed univariable linear regression with natural log(FC) or natural log(protein concentration [pg/ml]) as the dependent variables and natural log(FC viral N1 protein mRNA ) as the independent variable to understand associations with viral load, adjusting with age and sex as above. We performed sensitivity analyses of significant associations reported in Tables 1 and 2 . For each dependent biomarker with significant associations with infection status or viral load, we selected all other 19 All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted November 4, 2020. ; https://doi.org/10.1101/2020.10.30.20223545 doi: medRxiv preprint biomarkers reported to have significant correlations in Supplementary Table 2 as additional adjustment variables. Using these adjustment biomarkers one at a time, we assessed the impact on the estimates for infection status and viral load for each significant association reported in Tables 1 and 2. We assigned 50 as the C T value for undetectable mRNA. For undetectable proteins by bead based multiplex immunoassay, we assigned the minimum detection value. These assignments enable quantitative analysis without treating the values as missing. Results were similar when analysis was restricted to raw data derived from the 6 infected and 6 uninfected samples with the highest recovery of RNA. All calculations and statistical modeling were performed using the R statistical system. 89 All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted November 4, 2020. ; https://doi.org/10.1101/2020.10.30.20223545 doi: medRxiv preprint All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted November 4, 2020. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. 1, b. IFIT-1, c. IFIT-3 and d. Tetherin (BST-2) mRNAs. There were significant associations between increasing e. IFIT-3 mRNA and f. IFIT-3 protein fold changes and increasing viral N1 protein mRNA. Protein fold-change for IFIT-3 were measured using DDA mass spectrometry. Similar relationships were seen using viral E1 protein mRNA as in e. and f. In each panel, a-d, there are six infected and six non-infected status patients. All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted November 4, 2020. ; show log(pg/ml) of each protein as a function of viral detection. † Adjustment for sex was not significant, but patients had slightly decreased IFN-λ2,3 in response to SARS-CoV-2 infection for each additional year of age (OR = 0.99, 95% CI = 0.988-0.999, p = 0.048). 0.018 * Results show log(fold change of each mRNA) as a function of viral detection. † Adjustments for age and sex were not significant. ‡ BST-2 is also known as Tetherin. --MX-1 3.375 0.066 * χ 2 tests were applied to 2 × 2 tables of detection of protein vs detection of SARS-CoV-2 in all cases except for IFIT-1. There was detection of IFIT-1 in nearly all samples, however, they segregated into high level or low level detection, and the χ 2 test was applied to a 2 × 2 table of high detection of protein vs detection of SARS-CoV-2. For each result shown, there were n = 6 SARS-CoV-2 negative and n = 6 SARS-CoV-2 positive patients. † Not Detected. 33 All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted November 4, 2020. All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted November 4, 2020. All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. 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