Viral Resistance Analyses From the Remdesivir Phase 3 Adaptive COVID-19 Treatment Trial-1 (ACTT-1).

BACKGROUND: Remdesivir is approved for treatment of coronavirus disease 2019 (COVID-19) in nonhospitalized and hospitalized adult and pediatric patients. Here we present severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) resistance analyses from the phase 3 ACTT-1 randomized placebo-controlled trial conducted in adult participants hospitalized with COVID-19. METHODS: Swab samples were collected at baseline and longitudinally through day 29. SARS-CoV-2 genomes were sequenced using next-generation sequencing. Phenotypic analysis was conducted directly on participant virus isolates and/or using SARS-CoV-2 subgenomic replicons expressing mutations identified in the Nsp12 target gene. RESULTS: Among participants with both baseline and postbaseline sequencing data, emergent Nsp12 substitutions were observed in 12 of 31 (38.7%) and 12 of 30 (40.0%) participants in the remdesivir and placebo arms, respectively. No emergent Nsp12 substitutions in the remdesivir arm were observed in more than 1 participant. Phenotyping showed low to no change in susceptibility to remdesivir relative to wild-type Nsp12 reference for the substitutions tested: A16V (0.8-fold change in EC50), P323L + V792I (2.2-fold), C799F (2.5-fold), K59N (1.0-fold), and K59N + V792I (3.4-fold). CONCLUSIONS: The similar rate of emerging Nsp12 substitutions in the remdesivir and placebo arms and the minimal change in remdesivir susceptibility among tested substitutions support a high barrier to remdesivir resistance development in COVID-19 patients. Clinical Trials Registration. NCT04280705.

A Small Molecule RIG-I Agonist Serves as an Adjuvant to Induce Broad Multifaceted Influenza Virus Vaccine Immunity.

Retinoic acid-inducible gene I (RIG-I) is essential for activating host cell innate immunity to regulate the immune response against many RNA viruses. We previously identified that a small molecule compound, KIN1148, led to the activation of IFN regulatory factor 3 (IRF3) and served to enhance protection against influenza A virus (IAV) A/California/04/2009 infection. We have now determined direct binding of KIN1148 to RIG-I to drive expression of IFN regulatory factor 3 and NF-κB target genes, including specific immunomodulatory cytokines and chemokines. Intriguingly, KIN1148 does not lead to ATPase activity or compete with ATP for binding but activates RIG-I to induce antiviral gene expression programs distinct from type I IFN treatment. When administered in combination with a vaccine against IAV, KIN1148 induces both neutralizing Ab and IAV-specific T cell responses compared with vaccination alone, which induces comparatively poor responses. This robust KIN1148-adjuvanted immune response protects mice from lethal A/California/04/2009 and H5N1 IAV challenge. Importantly, KIN1148 also augments human CD8+ T cell activation. Thus, we have identified a small molecule RIG-I agonist that serves as an effective adjuvant in inducing noncanonical RIG-I activation for induction of innate immune programs that enhance adaptive immune protection of antiviral vaccination.

Duration of viral infectiousness and correlation with symptoms and diagnostic testing in non-hospitalized adults during acute SARS-CoV-2 infection: A longitudinal cohort study.

BACKGROUND: Guidelines for SARS-CoV-2 have relied on limited data on duration of viral infectiousness and correlation with COVID-19 symptoms and diagnostic testing. METHODS: We enrolled ambulatory adults with acute SARS-CoV-2 infection and performed serial measurements of COVID-19 symptoms, nasal swab viral RNA, nucleocapsid (N) and spike (S) antigens, and replication-competent SARS-CoV-2 by viral growth in culture. We determined average time from symptom onset to a first negative test result and estimated risk of infectiousness, as defined by positive viral growth in culture. RESULTS: Among 95 adults, median [interquartile range] time from symptom onset to first negative test result was 9 [5] days, 13 [6] days, 11 [4] days, and >19 days for S antigen, N antigen, culture growth, and viral RNA by RT-PCR, respectively. Beyond two weeks, virus growth and N antigen titers were rarely positive, while viral RNA remained detectable among half (26/51) of participants tested 21-30 days after symptom onset. Between 6-10 days from symptom onset, N antigen was strongly associated with culture positivity (relative risk=7.61, 95% CI: 3.01-19.22), whereas neither viral RNA nor symptoms were associated with culture positivity. During the 14 days following symptom onset, the presence of N antigen remained strongly associated (adjusted relative risk=7.66, 95% CI: 3.96-14.82) with culture positivity, regardless of COVID-19 symptoms. CONCLUSIONS: Most adults have replication-competent SARS-CoV-2 for 10-14 after symptom onset. N antigen testing is a strong predictor of viral infectiousness and may be a more suitable biomarker, rather than absence of symptoms or viral RNA, to discontinue isolation within two weeks from symptom onset.

Dynamics of SARS-CoV-2 VOC Neutralization and Novel mAb Reveal Protection against Omicron.

New variants of SARS-CoV-2 continue to emerge and evade immunity. We isolated SARS-CoV-2 temporally across the pandemic starting with the first emergence of the virus in the western hemisphere and evaluated the immune escape among variants. A clinic-to-lab viral isolation and characterization pipeline was established to rapidly isolate, sequence, and characterize SARS-CoV-2 variants. A virus neutralization assay was applied to quantitate humoral immunity from infection and/or vaccination. A panel of novel monoclonal antibodies was evaluated for antiviral efficacy. We directly compared all variants, showing that convalescence greater than 5 months post-symptom onset from ancestral virus provides little protection against SARS-CoV-2 variants. Vaccination enhances immunity against viral variants, except for Omicron BA.1, while a three-dose vaccine regimen provides over 50-fold enhanced protection against Omicron BA.1 compared to a two-dose. A novel Mab neutralizes Omicron BA.1 and BA.2 variants better than the clinically approved Mabs, although neither can neutralize Omicron BA.4 or BA.5. Thus, the need remains for continued vaccination-booster efforts, with innovation for vaccine and Mab improvement for broadly neutralizing activity. The usefulness of specific Mab applications links with the window of clinical opportunity when a cognate viral variant is present in the infected population.

Dynamics of SARS-CoV-2 VOC neutralization and novel mAb reveal protection against Omicron.

To evaluate SARS-CoV-2 variants we isolated SARS-CoV-2 temporally during the pandemic starting with first appearance of virus in the Western hemisphere near Seattle, WA, USA, and isolated each known major variant class, revealing the dynamics of emergence and complete take-over of all new cases by current Omicron variants. We assessed virus neutralization in a first-ever full comparison across variants and evaluated a novel monoclonal antibody (Mab). We found that convalescence greater than 5-months provides little-to-no protection against SARS-CoV-2 variants, vaccination enhances immunity against variants with the exception of Omicron BA.1, and paired testing of vaccine sera against ancestral virus compared to Omicron BA.1 shows that 3-dose vaccine regimen provides over 50-fold enhanced protection against Omicron BA.1 compared to a 2-dose regimen. We also reveal a novel Mab that effectively neutralizes Omicron BA.1 and BA.2 variants over clinically-approved Mabs. Our observations underscore the need for continued vaccination efforts, with innovation for vaccine and Mab improvement, for protection against variants of SARS-CoV-2. Summary: We isolated SARS-CoV-2 temporally starting with emergence of virus in the Western hemisphere. Neutralization analyses across all variant lineages show that vaccine-boost regimen provides protection against Omicron BA.1. We reveal a Mab that protects against Omicron BA.1 and BA.2 variants.

Immune Correlates of Protection From West Nile Virus Neuroinvasion and Disease.

BACKGROUND: A challenge to the design of improved therapeutic agents and prevention strategies for neuroinvasive infection and associated disease is the lack of known natural immune correlates of protection. A relevant model to study such correlates is offered by the Collaborative Cross (CC), a panel of recombinant inbred mouse strains that exhibit a range of disease manifestations upon infection. METHODS: We performed an extensive screen of CC-F1 lines infected with West Nile virus (WNV), including comprehensive immunophenotyping, to identify groups of lines that exhibited viral neuroinvasion or neuroinvasion with disease and lines that remained free of WNV neuroinvasion and disease. RESULTS: Our data reveal that protection from neuroinvasion and disease is multifactorial and that several immune outcomes can contribute. Immune correlates identified include decreased suppressive activity of regulatory T cells at steady state, which correlates with peripheral restriction of the virus. Further, a rapid contraction of WNV-specific CD8+ T cells in the brain correlated with protection from disease. CONCLUSIONS: These immune correlates of protection illustrate additional networks and pathways of the WNV immune response that cannot be observed in the C57BL/6 mouse model. Additionally, correlates of protection exhibited before infection, at baseline, provide insight into phenotypic differences in the human population that may predict clinical outcomes upon infection.

Interferon-stimulated genes: new platforms and computational approaches.

Interferon-stimulated genes (ISGs) are the effectors of interferon (IFN) actions and play major roles in innate immune defense against microbial infection. During virus infection, ISGs impart antiviral actions to control virus replication and spread but can also contribute to disease pathology if their expression is unchecked. Antiviral ISGs have been identified by a variety of biochemical, genetic, and virologic methods. New computational approaches are expanding and redefining ISGs as responders to a variety of stimuli beyond IFNs, including virus infection, stress, and other events that induce cytokines. These studies reveal that the expression of ISG subsets link to interferon regulatory factors (IRF)s, NF-kB, and other transcription factors that impart gene expression in specific cell types independently of IFNs, including stem cells and other cell types where ISGs are constitutively expressed. Here, we provide a broad overview of ISGs, define virus-induced genes (VSG)s, and discuss the application of computational approaches and bioinformatics platforms to evaluate the functional role of ISGs in epigenetics, immune programming, and vaccine responses.

RNA PAMPs as Molecular Tools for Evaluating RIG-I Function in Innate Immunity.

Pathogen recognition receptors (PRR)s and their cognate pathogen-associated molecular pattern (PAMP) represent the basis of innate immune activation and immune response induction driven by the host-pathogen interaction that occurs during microbial infection in humans and other animals. For RNA virus infection such as hepatitis C virus (HCV) and others, specific motifs within viral RNA mark it as nonself and visible to the host as a PAMP through interaction with RIG-I-like receptors including retinoic inducible gene-I (RIG-I). Here, we present methods for producing and using HCV PAMP RNA as a molecular tool to study RIG-I and its signaling pathway, both in vitro and in vivo, in innate immune regulation.

Oas1b-dependent Immune Transcriptional Profiles of West Nile Virus Infection in the Collaborative Cross.

The oligoadenylate-synthetase (Oas) gene locus provides innate immune resistance to virus infection. In mouse models, variation in the Oas1b gene influences host susceptibility to flavivirus infection. However, the impact of Oas variation on overall innate immune programming and global gene expression among tissues and in different genetic backgrounds has not been defined. We examined how Oas1b acts in spleen and brain tissue to limit West Nile virus (WNV) susceptibility and disease across a range of genetic backgrounds. The laboratory founder strains of the mouse Collaborative Cross (CC) (A/J, C57BL/6J, 129S1/SvImJ, NOD/ShiLtJ, and NZO/HlLtJ) all encode a truncated, defective Oas1b, whereas the three wild-derived inbred founder strains (CAST/EiJ, PWK/PhJ, and WSB/EiJ) encode a full-length OAS1B protein. We assessed disease profiles and transcriptional signatures of F1 hybrids derived from these founder strains. F1 hybrids included wild-type Oas1b (F/F), homozygous null Oas1b (N/N), and heterozygous offspring of both parental combinations (F/N and N/F). These mice were challenged with WNV, and brain and spleen samples were harvested for global gene expression analysis. We found that the Oas1b haplotype played a role in WNV susceptibility and disease metrics, but the presence of a functional Oas1b allele in heterozygous offspring did not absolutely predict protection against disease. Our results indicate that Oas1b status as wild-type or truncated, and overall Oas1b gene dosage, link with novel innate immune gene signatures that impact specific biological pathways for the control of flavivirus infection and immunity through both Oas1b-dependent and independent processes.

A small-molecule IRF3 agonist functions as an influenza vaccine adjuvant by modulating the antiviral immune response.

Vaccine adjuvants are essential to drive a protective immune response in cases where vaccine antigens are weakly immunogenic, where vaccine antigen is limited, or where an increase in potency is needed for a specific population, such as the elderly. To discover novel vaccine adjuvants, we used a high-throughput screen (HTS) designed to identify small-molecule agonists of the RIG-I-like receptor (RLR) pathway leading to interferon regulatory factor 3 (IRF3) activation. RLRs are a group of cytosolic pattern-recognition receptors that are essential for the recognition of viral nucleic acids during infection. Upon binding of viral nucleic acid ligands, the RLRs become activated and signal to transcription factors, including IRF3, to initiate an innate immune transcriptional program to control virus infection. Among our HTS hits were a series of benzothiazole compounds from which we designed the lead analog, KIN1148. KIN1148 induced dose-dependent IRF3 nuclear translocation and specific activation of IRF3-responsive promoters. Prime-boost immunization of mice with a suboptimal dose of a monovalent pandemic influenza split virus H1N1 A/California/07/2009 vaccine plus KIN1148 protected against a lethal challenge with mouse-adapted influenza virus (A/California/04/2009) and induced an influenza virus-specific IL-10 and Th2 response by T cells derived from lung and lung-draining lymph nodes. Prime-boost immunization with vaccine plus KIN1148, but not prime immunization alone, induced antibodies capable of inhibiting influenza virus hemagglutinin and neutralizing viral infectivity. Nevertheless, a single immunization with vaccine plus KIN1148 provided increased protection over vaccine alone and reduced viral load in the lungs after challenge. These findings suggest that protection was at least partially mediated by a cellular immune component and that the induction of Th2 and immunoregulatory cytokines by a KIN1148-adjuvanted vaccine may be particularly beneficial for ameliorating the immunopathogenesis that is associated with influenza viruses.

A Mouse Model of Chronic West Nile Virus Disease.

Infection with West Nile virus (WNV) leads to a range of disease outcomes, including chronic infection, though lack of a robust mouse model of chronic WNV infection has precluded identification of the immune events contributing to persistent infection. Using the Collaborative Cross, a population of recombinant inbred mouse strains with high levels of standing genetic variation, we have identified a mouse model of persistent WNV disease, with persistence of viral loads within the brain. Compared to lines exhibiting no disease or marked disease, the F1 cross CC(032x013)F1 displays a strong immunoregulatory signature upon infection that correlates with restraint of the WNV-directed cytolytic response. We hypothesize that this regulatory T cell response sufficiently restrains the immune response such that a chronic infection can be maintained in the CNS. Use of this new mouse model of chronic neuroinvasive virus will be critical in developing improved strategies to prevent prolonged disease in humans.

Identifying protective host gene expression signatures within the spleen during West Nile virus infection in the collaborative cross model.

Flaviviruses are hematophagous arthropod-viruses that pose global challenges to human health. Like Zika virus, West Nile Virus (WNV) is a flavivirus for which no approved vaccine exists [1]. The role host genetics play in early detection and response to WNV still remains largely unexplained. In order to capture the impact of genetic variation on innate immune responses, we studied gene expression following WNV infection using the collaborative cross (CC). The CC is a mouse genetics resource composed of hundreds of independently bred, octo-parental recombinant inbred mouse lines [2]. To accurately capture the host immune gene expression signatures of West Nile infection, we used the nanostring platform to evaluate expression in spleen tissue isolated from CC mice infected with WNV over a time course of 4, 7, and 12 days' post-infection [3]. Nanostring is a non-amplification based digital method to quantitate gene expression that uses color-coded molecular barcodes to detect hundreds of transcripts in a sample. Using this approach, we identified unique gene signatures in spleen tissue at days 4, 7, and 12 following WNV infection, which delineated distinct differences between asymptomatic and symptomatic CC lines. We also identified novel immune genes. Data was deposited into the Gene Expression Omnibus under accession GSE86000.

Transcriptional profiles of WNV neurovirulence in a genetically diverse Collaborative Cross population.

West Nile Virus (WNV) is a mosquito-transmitted virus from the Flaviviridae family that causes fever in 1 in 5 infected people. WNV can also become neuro-invasive and cross the blood-brain barrier leading to severe neurological symptoms in a subset of WNV infected individuals [1]. WNV neuro-invasion is believed to be influenced by a number of factors including host genetics. In order to explore these effects and recapitulate the complex immune genetic differences among individuals, we studied gene expression following WNV infection in the Collaborative Cross (CC) model. The CC is a mouse genetics resource composed of > 70 independently bred, octo-parental recombinant inbred mouse lines [2]. To identify the individual host gene expression signatures influencing protection or susceptibility to WNV disease and WNV neuroinvasion, we used the nanostring nsolver platform to quantify gene expression in brain tissue isolated from WNV-infected CC mice at days 4, 7 and 12 post-infection [3]. This nanostring technology provided a high throughput, non-amplification based mRNA quantitation method to detect immune genes involved in neuro-invasion. Data was deposited into the Gene Expression Omnibus (GEO) under accession GSE85999.

Targeting Innate Immunity for Antiviral Therapy through Small Molecule Agonists of the RLR Pathway.

UNLABELLED: The cellular response to virus infection is initiated when pathogen recognition receptors (PRR) engage viral pathogen-associated molecular patterns (PAMPs). This process results in induction of downstream signaling pathways that activate the transcription factor interferon regulatory factor 3 (IRF3). IRF3 plays a critical role in antiviral immunity to drive the expression of innate immune response genes, including those encoding antiviral factors, type 1 interferon, and immune modulatory cytokines, that act in concert to restrict virus replication. Thus, small molecule agonists that can promote IRF3 activation and induce innate immune gene expression could serve as antivirals to induce tissue-wide innate immunity for effective control of virus infection. We identified small molecule compounds that activate IRF3 to differentially induce discrete subsets of antiviral genes. We tested a lead compound and derivatives for the ability to suppress infections caused by a broad range of RNA viruses. Compound administration significantly decreased the viral RNA load in cultured cells that were infected with viruses of the family Flaviviridae, including West Nile virus, dengue virus, and hepatitis C virus, as well as viruses of the families Filoviridae (Ebola virus), Orthomyxoviridae (influenza A virus), Arenaviridae (Lassa virus), and Paramyxoviridae (respiratory syncytial virus, Nipah virus) to suppress infectious virus production. Knockdown studies mapped this response to the RIG-I-like receptor pathway. This work identifies a novel class of host-directed immune modulatory molecules that activate IRF3 to promote host antiviral responses to broadly suppress infections caused by RNA viruses of distinct genera. IMPORTANCE: Incidences of emerging and reemerging RNA viruses highlight a desperate need for broad-spectrum antiviral agents that can effectively control infections caused by viruses of distinct genera. We identified small molecule compounds that can selectively activate IRF3 for the purpose of identifying drug-like molecules that can be developed for the treatment of viral infections. Here, we report the discovery of a hydroxyquinoline family of small molecules that can activate IRF3 to promote cellular antiviral responses. These molecules can prophylactically or therapeutically control infection in cell culture by pathogenic RNA viruses, including West Nile virus, dengue virus, hepatitis C virus, influenza A virus, respiratory syncytial virus, Nipah virus, Lassa virus, and Ebola virus. Our study thus identifies a class of small molecules with a novel mechanism to enhance host immune responses for antiviral activity against a variety of RNA viruses that pose a significant health care burden and/or that are known to cause infections with high case fatality rates.

Genetic diversity in the collaborative cross model recapitulates human West Nile virus disease outcomes.

UNLABELLED: West Nile virus (WNV) is an emerging neuroinvasive flavivirus that now causes significant morbidity and mortality worldwide. The innate and adaptive immune responses to WNV infection have been well studied in C57BL/6J inbred mice, but this model lacks the variations in susceptibility, immunity, and outcome to WNV infection that are observed in humans, thus limiting its usefulness to understand the mechanisms of WNV infection and immunity dynamics. To build a model of WNV infection that captures human infection outcomes, we have used the Collaborative Cross (CC) mouse model. We show that this model, which recapitulates the genetic diversity of the human population, demonstrates diversity in susceptibility and outcomes of WNV infection observed in humans. Using multiple F1 crosses of CC mice, we identified a wide range of susceptibilities to infection, as demonstrated through differences in survival, clinical disease score, viral titer, and innate and adaptive immune responses in both peripheral tissues and the central nervous system. Additionally, we examined the Oas1b alleles in the CC mice and confirmed the previous finding that Oas1b plays a role in susceptibility to WNV; however, even within a given Oas1b allele status, we identified a wide range of strain-specific WNV-associated phenotypes. These results confirmed that the CC model is effective for identifying a repertoire of host genes involved in WNV resistance and susceptibility. The CC effectively models a wide range of WNV clinical, virologic, and immune phenotypes, thus overcoming the limitations of the traditional C57BL/6J model, allowing genetic and mechanistic studies of WNV infection and immunity in differently susceptible populations. IMPORTANCE: Mouse models of West Nile virus infection have revealed important details regarding the innate and adaptive immune responses to this emerging viral infection. However, traditional mouse models lack the genetic diversity present in human populations and therefore limit our ability to study various disease outcomes and immunologic mechanisms subsequent to West Nile virus infection. In this study, we used the Collaborative Cross mouse model to more effectively model the wide range of clinical, virologic, and immune phenotypes present upon West Nile virus infection in humans.

Pushing to a cure by harnessing innate immunity against hepatitis C virus.

Hepatitis C virus (HCV) causes 350,000 deaths and infects at least 3million people worldwide every year. Currently no vaccine has been developed. Direct-acting antiviral (DAA) drugs with high efficacy for suppressing HCV infection have recently been introduced into the clinic. While DAAs initially required combination therapy with type-1 interferon (IFN) administration for full efficacy and to avoid viral resistance to treatment, new DAA combinations show promise as an IFN-free regimen. However, IFN-free DAA therapy is in its infancy, still to be proven and today is cost-prohibitive for the patient. A major goal in HCV therapy to remove or replace IFN with DAAs or an alternative therapeutic to render virologic response with continued virus sensitivity to DAAs, thus facilitating a cure for infection. Recent advances in our understanding of innate immune responses to HCV have identified new therapeutic targets to combat HCV infection. We discuss how the targeting of innate immune response factors can be harnessed with DAAs to produce new generations of DAA-based HCV therapeutics. This article forms part of a symposium in Antiviral Research on "Hepatitis C: next steps toward global eradication."

Systems biology analyses to define host responses to HCV infection and therapy.

While 170 million people worldwide are chronically infected with HCV, the response rate to the current treatment regimens of pegylated IFN-α (IFN) in combination with ribavirin is only approximately 55 % of all HCV patients undergoing therapy. This IFN-based therapy is now slated to serve as the backbone for future combination therapeutics involving direct-acting antiviral compounds, including HCV protease inhibitors, viral polymerase inhibitors, and other small molecules. It is essential that the application of IFN be improved for overall enhancement of therapy outcome to effectively cure HCV infection. Systems approaches, including genomics and network modeling, are particularly powerful tools that are now being used to dissect the underlying mechanisms of successful or failed treatment response in an effort to design improved IFN-based therapeutic regimens. Furthermore, systems applications can be used to define virus-host interactions and map their variation within viral and host genomes, leading to identification of targets for novel therapy strategies. Using these approaches, we have defined distinct hepatic expression and tissue distribution of innate immune signaling molecules and gene networks that associate with IFN-based treatment outcome for HCV infection. This chapter will focus on using systems approaches to understand the host response to both HCV infection and therapy to drive the development of improved HCV therapeutics.

RIG-I like receptors in antiviral immunity and therapeutic applications.

The RNA helicase family of RIG-I-like receptors (RLRs) is a key component of host defense mechanisms responsible for detecting viruses and triggering innate immune signaling cascades to control viral replication and dissemination. As cytoplasm-based sensors, RLRs recognize foreign RNA in the cell and activate a cascade of antiviral responses including the induction of type I interferons, inflammasome activation, and expression of proinflammatory cytokines and chemokines. This review provides a brief overview of RLR function, ligand interactions, and downstream signaling events with an expanded discussion on the therapeutic potential of targeting RLRs for immune stimulation and treatment of virus infection.

Integrated proteomic analysis of human cancer cells and plasma from tumor bearing mice for ovarian cancer biomarker discovery.

BACKGROUND: The complexity of the human plasma proteome represents a substantial challenge for biomarker discovery. Proteomic analysis of genetically engineered mouse models of cancer and isolated cancer cells and cell lines provide alternative methods for identification of potential cancer markers that would be detectable in human blood using sensitive assays. The goal of this work is to evaluate the utility of an integrative strategy using these two approaches for biomarker discovery. METHODOLOGY/PRINCIPAL FINDINGS: We investigated a strategy that combined quantitative plasma proteomics of an ovarian cancer mouse model with analysis of proteins secreted or shed by human ovarian cancer cells. Of 106 plasma proteins identified with increased levels in tumor bearing mice, 58 were also secreted or shed from ovarian cancer cells. The remainder consisted primarily of host-response proteins. Of 25 proteins identified in the study that were assayed, 8 mostly secreted proteins common to mouse plasma and human cancer cells were significantly upregulated in a set of plasmas from ovarian cancer patients. Five of the eight proteins were confirmed to be upregulated in a second independent set of ovarian cancer plasmas, including in early stage disease. CONCLUSIONS/SIGNIFICANCE: Integrated proteomic analysis of cancer mouse models and human cancer cell populations provides an effective approach to identify potential circulating protein biomarkers.