Species-specific quantification of circulating ebolavirus burden using VP40-derived peptide variants.

Six ebolavirus species are reported to date, including human pathogens Bundibugyo virus (BDBV), Ebola virus (EBOV), Sudan virus (SUDV), and Taï Forest virus (TAFV); non-human pathogen Reston virus (RESTV); and the plausible Bombali virus (BOMV). Since there are differences in the disease severity caused by different species, species identification and viral burden quantification are critical for treating infected patients timely and effectively. Here we developed an immunoprecipitation-coupled mass spectrometry (IP-MS) assay for VP40 antigen detection and quantification. We carefully selected two regions of VP40, designated as peptide 8 and peptide12 from the protein sequence that showed minor variations among Ebolavirus species through MS analysis of tryptic peptides and antigenicity prediction based on available bioinformatic tools, and generated high-quality capture antibodies pan-specific for these variant peptides. We applied this assay to human plasma spiked with recombinant VP40 protein from EBOV, SUDV, and BDBV and virus-like particles (VLP), as well as EBOV infected NHP plasma. Sequence substitutions between EBOV and SUDV, the two species with highest lethality, produced affinity variations of 2.6-fold for p8 and 19-fold for p12. The proposed IP-MS assay differentiates four of the six known EBV species in one assay, through a combination of p8 and p12 data. The IP-MS assay limit of detection (LOD) using multiple reaction monitoring (MRM) as signal readout was determined to be 28 ng/mL and 7 ng/mL for EBOV and SUDV respectively, equivalent to ~1.625-6.5×105 Geq/mL, and comparable to the LOD of lateral flow immunoassays currently used for Ebola surveillance. The two peptides of the IP-MS assay were also identified by their tandem MS spectra using a miniature MALDI-TOF MS instrument, greatly increasing the feasibility of high specificity assay in a decentralized laboratory.

Characterization of Citrullination Sites in Neutrophils and Mast Cells Activated by Ionomycin via Integration of Mass Spectrometry and Machine Learning.

Citrullination is an important post-translational modification implicated in many diseases including rheumatoid arthritis (RA), Alzheimer's disease, and cancer. Neutrophil and mast cells have different expression profiles for protein-arginine deiminases (PADs), and ionomycin-induced activation makes them an ideal cellular model to study proteins susceptible to citrullination. We performed high-resolution mass spectrometry and stringent data filtration to identify citrullination sites in neutrophil and mast cells treated with and without ionomycin. We identified a total of 833 validated citrullination sites on 395 proteins. Several of these citrullinated proteins are important components of pathways involved in innate immune responses. Using this benchmark primary sequence data set, we developed machine learning models to predict citrullination in neutrophil and mast cell proteins. We show that our models predict citrullination likelihood with 0.735 and 0.766 AUCs (area under the receiver operating characteristic curves), respectively, on independent validation sets. In summary, this study provides the largest number of validated citrullination sites in neutrophil and mast cell proteins. The use of our novel motif analysis approach to predict citrullination sites will facilitate the discovery of novel protein substrates of protein-arginine deiminases (PADs), which may be key to understanding immunopathologies of various diseases.

Development of a Parallel Reaction Monitoring Mass Spectrometry Assay for the Detection of SARS-CoV-2 Spike Glycoprotein and Nucleoprotein.

There is an urgent need for robust and high-throughput methods for SARS-CoV-2 detection in suspected patient samples to facilitate disease management, surveillance, and control. Although nucleic acid detection methods such as reverse transcription polymerase chain reaction (RT-PCR) are the gold standard, during the current pandemic, the deployment of RT-PCR tests has been extremely slow, and key reagents such as PCR primers and RNA extraction kits are at critical shortages. Rapid point-of-care viral antigen detection methods have been previously employed for the diagnosis of respiratory viruses such as influenza and respiratory syncytial viruses. Therefore, the direct detection of SARS-CoV-2 viral antigens in patient samples could also be used for diagnosis of active infection, and alternative methodologies for specific and sensitive viral protein detection should be explored. Targeted mass spectrometry techniques have enabled the identification and quantitation of a defined subset of proteins/peptides at single amino acid resolution with attomole level sensitivity and high reproducibility. Herein, we report a targeted mass spectrometry assay for the detection of SARS-CoV-2 spike protein and nucleoprotein in a relevant biological matrix. Recombinant full-length spike protein and nucleoprotein were digested and proteotypic peptides were selected for parallel reaction monitoring (PRM) quantitation using a high-resolution Orbitrap instrument. A spectral library, which contained seven proteotypic peptides (four from spike protein and three from nucleoprotein) and the top three to four transitions, was generated and evaluated. From the original spectral library, we selected two best performing peptides for the final PRM assay. The assay was evaluated using mock test samples containing inactivated SARS-CoV-2 virions, added to in vitro derived mucus. The PRM assay provided a limit of detection of ∼200 attomoles and a limit of quantitation of ∼ 390 attomoles. Extrapolating from the test samples, the projected titer of virus particles necessary for the detection of SARS-CoV-2 spike and nucleoprotein detection was approximately 2 × 105 viral particles/mL, making it an attractive alternative to RT-PCR assays. Potentially, mass spectrometry-based methods for viral antigen detection may deliver higher throughput and could serve as a complementary diagnostic tool to RT-PCR. Furthermore, this assay could be used to evaluate the presence of SARS-CoV-2 in archived or recently collected biological fluids, in vitro-derived research materials, and wastewater samples.

Early detection of Ebola virus proteins in peripheral blood mononuclear cells from infected mice.

BACKGROUND: Detection of viral ribo-nucleic acid (RNA) via real-time polymerase chain reaction (RT-PCR) is the gold standard for the detection of Ebola virus (EBOV) during acute infection. However, the earliest window for viral RNA detection in blood samples is 48-72 h post-onset of symptoms. Therefore, efforts to develop additional orthogonal assays using complementary immunological and serological technologies are still needed to provide simplified methodology for field diagnostics. Furthermore, unlike RT-PCR tests, immunoassays that target viral proteins and/or early host responses are less susceptible to sequence erosion due to viral genetic drift. Although virus is shed into the bloodstream from infected cells, the wide dynamic range of proteins in blood plasma makes this a difficult sample matrix for the detection of low-abundant viral proteins. We hypothesized that the isolation of peripheral blood mononuclear cells (PBMCs), which are the first cellular targets of the Ebola virus (EBOV), may provide an enriched source of viral proteins. METHODS: A mouse infection model that employs a mouse-adapted EBOV (MaEBOV) was chosen as a proof-of-principal experimental paradigm to determine if viral proteins present in PBMCs can help diagnose EBOV infection pre-symptomatically. We employed a liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) platform to provide both high sensitivity and specificity for the detection and relative quantitation of viral proteins in PBMCs collected during MaEBOV infection. Blood samples pooled from animals at the post-infection time-points were used to determine the viral load by RT-PCR and purify PBMCs. RESULTS: Using quantitative LC-MS/MS, we detected two EBOV proteins (vp40 and nucleoprotein) in samples collected on Day 2 post-infection, which was also the first day of detectable viremia via RT-PCR. These results were confirmed via western blot which was performed on identical PBMC lysates from each post-infection time point. CONCLUSIONS: While mass spectrometry is not currently amenable to field diagnostics, these results suggest that viral protein enrichment in PBMCs in tandem with highly sensitive immunoassays platforms, could lead to the development of a rapid, high-throughput diagnostic platform for pre-symptomatic detection of EBOV infection.

Quantification of circulating Mycobacterium tuberculosis antigen peptides allows rapid diagnosis of active disease and treatment monitoring.

Tuberculosis (TB) is a major global health threat, resulting in an urgent unmet need for a rapid, non-sputum-based quantitative test to detect active Mycobacterium tuberculosis (Mtb) infections in clinically diverse populations and quickly assess Mtb treatment responses for emerging drug-resistant strains. We have identified Mtb-specific peptide fragments and developed a method to rapidly quantify their serum concentrations, using antibody-labeled and energy-focusing porous discoidal silicon nanoparticles (nanodisks) and high-throughput mass spectrometry (MS) to enhance sensitivity and specificity. NanoDisk-MS diagnosed active Mtb cases with high sensitivity and specificity in a case-control study with cohorts reflecting the complexity of clinical practice. Similar robust sensitivities were obtained for cases of culture-positive pulmonary TB (PTB; 91.3%) and extrapulmonary TB (EPTB; 92.3%), and the sensitivities obtained for culture-negative PTB (82.4%) and EPTB (75.0%) in HIV-positive patients significantly outperformed those reported for other available assays. NanoDisk-MS also exhibited high specificity (87.1-100%) in both healthy and high-risk groups. Absolute quantification of serum Mtb antigen concentration was informative in assessing responses to antimycobacterial treatment. Thus, a NanoDisk-MS assay approach could significantly improve the diagnosis and management of active TB cases, and perhaps other infectious diseases as well.

Characterization of the plasma proteome of nonhuman primates during Ebola virus disease or melioidosis: a host response comparison.

BACKGROUND: In-depth examination of the plasma proteomic response to infection with a wide variety of pathogens can assist in the development of new diagnostic paradigms, while providing insight into the interdependent pathogenic processes which encompass a host's immunological and physiological responses. Ebola virus (EBOV) causes a highly lethal infection termed Ebola virus disease (EVD) in primates and humans. The Gram negative non-spore forming bacillus Burkholderia pseudomallei (Bp) causes melioidosis in primates and humans, characterized by severe pneumonia with high mortality. We sought to examine the host response to infection with these two bio-threat pathogens using established animal models to provide information on the feasibility of pre-symptomatic diagnosis, since the induction of host molecular signaling networks can occur before clinical presentation and pathogen detection. METHODS: Herein we report the quantitative proteomic analysis of plasma collected at various times of disease progression from 10 EBOV-infected and 5 Bp-infected nonhuman primates (NHP). Our strategy employed high resolution LC-MS/MS and a peptide-tagging approach for relative protein quantitation. In each infection type, for all proteins with > 1.3 fold abundance change at any post-infection time point, a direct comparison was made with levels obtained from plasma collected daily from 5 naïve rhesus macaques, to determine the fold changes that were significant, and establish the natural variability of abundance for endogenous plasma proteins. RESULTS: A total of 41 plasma proteins displayed significant alterations in abundance during EBOV infection, and 28 proteins had altered levels during Bp infection, when compared to naïve NHPs. Many major acute phase proteins quantitated displayed similar fold-changes between the two infection types but exhibited different temporal dynamics. Proteins related to the clotting cascade, immune signaling and complement system exhibited significant differential abundance during infection with EBOV or Bp, indicating a specificity of the response. CONCLUSIONS: These results advance our understanding of the global plasma proteomic response to EBOV and Bp infection in relevant primate models for human disease and provide insight into potential innate immune response differences between viral and bacterial infections.

Countering Zika Virus: The USAMRIID Response.

The United States Army Medical Research Institute of Infectious Diseases (USAMRIID) possesses an array of expertise in diverse capabilities for the characterization of emerging infectious diseases from the pathogen itself to human or animal infection models. The recent Zika virus (ZIKV) outbreak was a challenge and an opportunity to put these capabilities to work as a cohesive unit to quickly respond to a rapidly developing threat. Next-generation sequencing was used to characterize virus stocks and to understand the introduction and spread of ZIKV in the United States. High Content Imaging was used to establish a High Content Screening process to evaluate antiviral therapies. Functional genomics was used to identify critical host factors for ZIKV infection. An animal model using the temporal blockade of IFN-I in immunocompetent laboratory mice was investigated in conjunction with Positron Emission Tomography to study ZIKV. Correlative light and electron microscopy was used to examine ZIKV interaction with host cells in culture and infected animals. A quantitative mass spectrometry approach was used to examine the protein and metabolite type or concentration changes that occur during ZIKV infection in blood, cells, and tissues. Multiplex fluorescence in situ hybridization was used to confirm ZIKV replication in mouse and NHP tissues. The integrated rapid response approach developed at USAMRIID presented in this review was successfully applied and provides a new template pathway to follow if a new biological threat emerges. This streamlined approach will increase the likelihood that novel medical countermeasures could be rapidly developed, evaluated, and translated into the clinic.

High Infection Rates for Adult Macaques after Intravaginal or Intrarectal Inoculation with Zika Virus.

Unprotected sexual intercourse between persons residing in or traveling from regions with Zika virus transmission is a risk factor for infection. To model risk for infection after sexual intercourse, we inoculated rhesus and cynomolgus macaques with Zika virus by intravaginal or intrarectal routes. In macaques inoculated intravaginally, we detected viremia and virus RNA in 50% of macaques, followed by seroconversion. In macaques inoculated intrarectally, we detected viremia, virus RNA, or both, in 100% of both species, followed by seroconversion. The magnitude and duration of infectious virus in the blood of macaques suggest humans infected with Zika virus through sexual transmission will likely generate viremias sufficient to infect competent mosquito vectors. Our results indicate that transmission of Zika virus by sexual intercourse might serve as a virus maintenance mechanism in the absence of mosquito-to-human transmission and could increase the probability of establishment and spread of Zika virus in regions where this virus is not present.

CCL5-CCR5 interactions modulate metabolic events during tumor onset to promote tumorigenesis.

BACKGROUND: In earlier studies we have shown that CCL5 activation of CCR5 induces the proliferation and survival of breast cancer cells in a mechanistic target of rapamycin (mTOR)-dependent manner and that this is in part due to CCR5-mediated increases in glycolytic metabolism. METHODS: Using the MDA-MB-231 triple negative human breast cancer cell line and mouse mammary tumor virus - polyomavirus middle T-antigen (MMTV-PyMT) mouse primary breast cancer cells, we conducted in vivo tumor transplant experiments to examine the effects of CCL5-CCR5 interactions in the context of regulating tumor metabolism. Additionally, we employed Matrix-Assisted Laser Desorption/Ionization Fourier Transform Ion Cyclotron Resonance Mass Spectrometry imaging (MALDI-FTICR-MSI) to evaluate tumor utilization of cellular metabolites. RESULTS: We provide evidence that, in the absence of CCR5, the early events associated with rapid tumor growth in the MMTV-PyMT mouse model of spontaneous breast cancer development, are diminished, as demonstrated by a delay in tumor onset. In tumor transplant studies into immunocompromised mice we identify a direct correlation between reduced tumor proliferation and decreased metabolic activity, specifically associated with tumor expression of CCR5. The reduction in tumorigenesis is accompanied by decreases in glucose uptake, glucose transporter-1 (GLUT-1) cell surface expression, intracellular ATP and lactate levels, as well as reduced CCL5 production. Using MALDI-FTICR-MS, we show that the rapid early tumor growth of CCR5+/+ triple negative breast cancer cells in vivo is attributable to increased levels of glycolytic intermediates required for anabolic processes, in contrast to the slower growth rate of their corresponding CCR5-/- cells, that exhibit reduced glycolytic metabolism. CONCLUSIONS: These findings suggest that CCL5-CCR5 interactions in the tumor microenvironment modulate metabolic events during tumor onset to promote tumorigenesis.

Development of a liquid chromatography high resolution mass spectrometry method for the quantitation of viral envelope glycoprotein in Ebola virus-like particle vaccine preparations.

BACKGROUND: Ebola virus like particles (EBOV VLPs, eVLPs), are produced by expressing the viral transmembrane glycoprotein (GP) and structural matrix protein VP40 in mammalian cells. When expressed, these proteins self-assemble and bud from 'host' cells displaying morphology similar to infectious virions. Several studies have shown that rodents and non-human primates vaccinated with eVLPs are protected from lethal EBOV challenge. The mucin-like domain of envelope glycoprotein GP1 serves as the major target for a productive humoral immune response. Therefore GP1 concentration is a critical quality attribute of EBOV vaccines and accurate measurement of the amount of GP1 present in eVLP lots is crucial to understanding variability in vaccine efficacy. METHODS: After production, eVLPs are characterized by determining total protein concentration and by western blotting, which only provides semi-quantitative information for GP1. Therefore, a liquid chromatography high resolution mass spectrometry (LC-HRMS) approach for accurately measuring GP1 concentration in eVLPs was developed. The method employs an isotope dilution strategy using four target peptides from two regions of the GP1 protein. Purified recombinant GP1 was generated to serve as an assay standard. GP1 quantitation in 5 eVLP lots was performed on an LTQ-Orbitrap Elite and the final quantitation was derived by comparing the relative response of 200 fmol AQUA peptide standards to the analyte response at 4 ppm. RESULTS: Conditions were optimized to ensure complete tryptic digestion of eVLP, however, persistent missed cleavages were observed in target peptides. Additionally, N-terminal truncated forms of the GP1 protein were observed in all eVLP lots, making peptide selection crucial. The LC-HRMS strategy resulted in quantitation of GP1 with a lower limit of quantitation of 1 fmol and an average percent coefficient of variation (CV) of 7.6 %. Unlike western blot values, the LC-HRMS quantitation of GP1 in 5 eVLP vaccine lots exhibited a strong linear relationship (positive correlation) with survival (after EBOV challenge) in mice. CONCLUSIONS: This method provides a means to rapidly determine eVLP batch quality based upon quantitation of antigenic GP1. By monitoring variability in GP1 content, the eVLP production process can be optimized, and the total amount of GP1 needed to confer protection accurately determined.

Heat fixation inactivates viral and bacterial pathogens and is compatible with downstream MALDI mass spectrometry tissue imaging.

BACKGROUND: Tissue samples should be fixed and permanently stabilized as soon as possible ex-vivo to avoid variations in proteomic content. Tissues collected from studies involving infectious microorganisms, must face the additional challenge of pathogen inactivation before downstream proteomic analysis can be safely performed. Heat fixation using the Denator Stabilizor System (Gothenburg, Sweden) utilizes conductive heating, under a mild vacuum, to rapidly eliminate enzymatic degradation in tissue samples. Although many studies have reported on the ability of this method to stop proteolytic degradation and other sample changes immediately and permanently, pathogen inactivation has not been studied. RESULTS: We examined the ability of the heat fixation workflow to inactivate bacterial and viral pathogens and the suitability of this tissue for Matrix Assisted Laser Desorption Ionization mass spectrometry imaging (MALDI-MSI). Mice were infected with viral or bacterial pathogens representing two strains of Venezuelan Equine Encephalitis virus (VEEV) and two strains of Burkholderia. Additionally, a tissue mimetic model was employed using Escherichia, Klebsiella and Acinetobacter isolates. Infected tissue samples harvested from each animal or mimetic model were sectioned in half. One half was heat fixed and the other remained untreated. Lysates from each sample were checked for organism viability by performing plaque (infectivity) assays or plating on nutrient agar for colony forming unit (CFU) calculation. Untreated infected control tissue demonstrated the presence of each viable pathogen by positive plaque or colony formation, whereas heat fixation resulted in complete inactivation of both the viral and bacterial pathogens. MALDI-MSI images produced from heat fixed tissue were reflective of molecular distributions within brain, spleen and lung tissue structures. CONCLUSIONS: We conclude that heat fixation inactivates viral and bacterial pathogens and is compatible with proteomic analysis by MALDI-MSI. This treatment will enable the use of infected tissue from studies performed in bio-safety level 3 laboratories with VEEV and Burkholderia to be safely used for proteomic, small molecule drug detection, and imaging mass spectrometry analysis.

MALDI imaging mass spectrometry profiling of proteins and lipids in clear cell renal cell carcinoma.

Reducing the incidence and mortality rates for clear cell renal cell carcinoma (ccRCC) remains a significant clinical challenge with poor 5-year survival rates. A unique tissue cohort was assembled of matched ccRCC and distal nontumor tissues (n = 20) associated with moderate risk of disease progression, half of these from individuals who progressed to metastatic disease and the other half who remained disease free. These tissues were used for MALDI imaging MS profiling of proteins in the 2-20 kDa range, resulting in a panel of 108 proteins that had potential disease-specific expression patterns. Protein lysates from the same tissues were analyzed by MS/MS, resulting in identification of 56 proteins of less than 20 kDa molecular weight. The same tissues were also used for global lipid profiling analysis by MALDI-FT-ICR MS. From the cumulative protein and lipid expression profile data, a refined panel of 26 proteins and 39 lipid species was identified that could either distinguish tumor from nontumor tissues, or tissues from recurrent disease progressors from nonrecurrent disease individuals. This approach has the potential to not only improve prognostic assessment and enhance postoperative surveillance, but also to inform on the underlying biology of ccRCC progression.

Nucleocapsid protein-specific monoclonal antibodies protect mice against Crimean-Congo hemorrhagic fever virus.

Crimean-Congo hemorrhagic fever virus (CCHFV) is a WHO priority pathogen. Antibody-based medical countermeasures offer an important strategy to mitigate severe disease caused by CCHFV. Most efforts have focused on targeting the viral glycoproteins. However, glycoproteins are poorly conserved among viral strains. The CCHFV nucleocapsid protein (NP) is highly conserved between CCHFV strains. Here, we investigate the protective efficacy of a CCHFV monoclonal antibody targeting the NP. We find that an anti-NP monoclonal antibody (mAb-9D5) protected female mice against lethal CCHFV infection or resulted in a significant delay in mean time-to-death in mice that succumbed to disease compared to isotype control animals. Antibody protection is independent of Fc-receptor functionality and complement activity. The antibody bound NP from several CCHFV strains and exhibited robust cross-protection against the heterologous CCHFV strain Afg09-2990. Our work demonstrates that the NP is a viable target for antibody-based therapeutics, providing another direction for developing immunotherapeutics against CCHFV.

MALDI tissue imaging: from biomarker discovery to clinical applications.

Matrix-assisted laser desorption ionization (MALDI) imaging mass spectrometry (IMS) is a powerful tool for the generation of multidimensional spatial expression maps of biomolecules directly from a tissue section. From a clinical proteomics perspective, this method correlates molecular detail to histopathological changes found in patient-derived tissues, enhancing the ability to identify candidates for disease biomarkers. The unbiased analysis and spatial mapping of a variety of molecules directly from clinical tissue sections can be achieved through this method. Conversely, targeted IMS, by the incorporation of laser-reactive molecular tags onto antibodies, aptamers, and other affinity molecules, enables analysis of specific molecules or a class of molecules. In addition to exploring tissue during biomarker discovery, the integration of MALDI-IMS methods into existing clinical pathology laboratory practices could prove beneficial to diagnostics. Querying tissue for the expression of specific biomarkers in a biopsy is a critical component in clinical decision-making and such markers are a major goal of translational research. An important challenge in cancer diagnostics will be to assay multiple parameters in a single slide when tissue quantities are limited. The development of multiplexed assays that maximize the yield of information from a small biopsy will help meet a critical challenge to current biomarker research. This review focuses on the use of MALDI-IMS in biomarker discovery and its potential as a clinical diagnostic tool with specific reference to our application of this technology to prostate cancer.

Metabolomic Profiling of Human Urine Samples Using LC-TIMS-QTOF Mass Spectrometry.

The identification of metabolites in biological samples is challenging due to their chemical and structural diversity. Ion mobility spectrometry (IMS) separates ionized molecules based on their mobility in a carrier buffer gas giving information about the ionic shape by measuring the rotationally averaged collision cross-section (CCS) value. This orthogonal descriptor, in combination with the m/z, isotopic pattern distribution, and MS/MS spectrum, has the potential to improve the identification of molecular molecules in complex mixtures. Urine metabolomics can reveal metabolic differences, which arise as a result of a specific disease or in response to therapeutic intervention. It is, however, complicated by the presence of metabolic breakdown products derived from a wide range of lifestyle and diet-related byproducts, many of which are poorly characterized. In this study, we explore the use of trapped ion mobility spectrometry (TIMS) via LC parallel accumulation with serial fragmentation (PASEF) for urine metabolomics. A total of 362 urine metabolites were characterized from 80 urine samples collected from healthy volunteers using untargeted metabolomics employing HILIC and RP chromatography. Additionally, three analytes (Trp, Phe, and Tyr) were selected for targeted quantification. Both the untargeted and targeted data was highly reproducible and reported CCS measurements for identified metabolites were robust in the presence of the urine matrix. A comparison of CCS values among different laboratories was also conducted, showing less than 1.3% ΔCCS values across different platforms. This is the first report of a human urine metabolite database compiled with CCS values experimentally acquired using an LC-PASEF TIMS-qTOF platform.

Paracrine IFN Response Limits ZIKV Infection in Human Sertoli Cells.

Zika virus (ZIKV) is unique among mosquito-borne flaviviruses in its ability to be sexually transmitted. The testes have been implicated as sites of long-term ZIKV replication, and our previous studies have identified Sertoli cells (SC), the nurse cells of the seminiferous epithelium that govern spermatogenesis, as major targets of ZIKV infection. To improve our understanding of the interaction of ZIKV with human SC, we analyzed ZIKV-induced proteome changes in these cells using high-throughput liquid chromatography-tandem mass spectrometry (LC-MS/MS). Our data demonstrated that interferon (IFN) signaling was the most significantly enriched pathway and the antiviral proteins MX1 and IFIT1 were among the top upregulated proteins in SC following ZIKV infection. The dynamic between IFN response and ZIKV infection kinetics in SC remains unclear, therefore we further determined whether MX1 and IFIT1 serve as antiviral effectors against ZIKV. We found that increased levels of MX1 at the later time points of infection coincided with diminished ZIKV infection while the silencing of MX1 and IFIT1 enhanced peak ZIKV propagation in SC. Furthermore, although IFN-I exposure was found to significantly hinder ZIKV replication in SC, IFN response was attenuated in these cells as compared to other cell types. The data in this study highlight IFN-I as a driver of the antiviral state that limits ZIKV infection in SC and suggests that MX1 and IFIT1 function as antiviral effectors against ZIKV in SC. Collectively, this study provides important biological insights into the response of SC to ZIKV infection and the ability of the virus to persist in the testes.

Impact of Toll-Like Receptor-Specific Agonists on the Host Immune Response to the Yersinia pestis Plague rF1V Vaccine.

Relatively recent advances in plague vaccinology have produced the recombinant fusion protein F1-V plague vaccine. This vaccine has been shown to readily protect mice from both bubonic and pneumonic plague. The protection afforded by this vaccine is solely based upon the immune response elicited by the F1 or V epitopes expressed on the F1-V fusion protein. Accordingly, questions remain surrounding its efficacy against infection with non-encapsulated (F1-negative) strains. In an attempt to further optimize the F1-V elicited immune response and address efficacy concerns, we examined the inclusion of multiple toll-like receptor agonists into vaccine regimens. We examined the resulting immune responses and also any protection afforded to mice that were exposed to aerosolized Yersinia pestis. Our data demonstrate that it is possible to further augment the F1-V vaccine strategy in order to optimize and augment vaccine efficacy.

Proteomic Analysis of Non-human Primate Peripheral Blood Mononuclear Cells During Burkholderia mallei Infection Reveals a Role of Ezrin in Glanders Pathogenesis.

Burkholderia mallei, the causative agent of glanders, is a gram-negative intracellular bacterium. Depending on different routes of infection, the disease is manifested by pneumonia, septicemia, and chronic infections of the skin. B. mallei poses a serious biological threat due to its ability to infect via aerosol route, resistance to multiple antibiotics and to date there are no US Food and Drug Administration (FDA) approved vaccines available. Induction of innate immunity, inflammatory cytokines and chemokines following B. mallei infection, have been observed in in vitro and small rodent models; however, a global characterization of host responses has never been systematically investigated using a non-human primate (NHP) model. Here, using a liquid chromatography-tandem mass spectrometry (LC-MS/MS) approach, we identified alterations in expression levels of host proteins in peripheral blood mononuclear cells (PBMCs) originating from naïve rhesus macaques (Macaca mulatta), African green monkeys (Chlorocebus sabaeus), and cynomolgus macaques (Macaca fascicularis) exposed to aerosolized B. mallei. Gene ontology (GO) analysis identified several statistically significant overrepresented biological annotations including complement and coagulation cascade, nucleoside metabolic process, vesicle-mediated transport, intracellular signal transduction and cytoskeletal protein binding. By integrating an LC-MS/MS derived proteomics dataset with a previously published B. mallei host-pathogen interaction dataset, a statistically significant predictive protein-protein interaction (PPI) network was constructed. Pharmacological perturbation of one component of the PPI network, specifically ezrin, reduced B. mallei mediated interleukin-1ß (IL-1ß). On the contrary, the expression of IL-1ß receptor antagonist (IL-1Ra) was upregulated upon pretreatment with the ezrin inhibitor. Taken together, inflammasome activation as demonstrated by IL-1ß production and the homeostasis of inflammatory response is critical during the pathogenesis of glanders. Furthermore, the topology of the network reflects the underlying molecular mechanism of B. mallei infections in the NHP model.

A Bayesian network approach to feature selection in mass spectrometry data.

BACKGROUND: Time-of-flight mass spectrometry (TOF-MS) has the potential to provide non-invasive, high-throughput screening for cancers and other serious diseases via detection of protein biomarkers in blood or other accessible biologic samples. Unfortunately, this potential has largely been unrealized to date due to the high variability of measurements, uncertainties in the distribution of proteins in a given population, and the difficulty of extracting repeatable diagnostic markers using current statistical tools. With studies consisting of perhaps only dozens of samples, and possibly hundreds of variables, overfitting is a serious complication. To overcome these difficulties, we have developed a Bayesian inductive method which uses model-independent methods of discovering relationships between spectral features. This method appears to efficiently discover network models which not only identify connections between the disease and key features, but also organizes relationships between features--and furthermore creates a stable classifier that categorizes new data at predicted error rates. RESULTS: The method was applied to artificial data with known feature relationships and typical TOF-MS variability introduced, and was able to recover those relationships nearly perfectly. It was also applied to blood sera data from a 2004 leukemia study, and showed high stability of selected features under cross-validation. Verification of results using withheld data showed excellent predictive power. The method showed improvement over traditional techniques, and naturally incorporated measurement uncertainties. The relationships discovered between features allowed preliminary identification of a protein biomarker which was consistent with other cancer studies and later verified experimentally. CONCLUSIONS: This method appears to avoid overfitting in biologic data and produce stable feature sets in a network model. The network structure provides additional information about the relationships among features that is useful to guide further biochemical analysis. In addition, when used to classify new data, these feature sets are far more consistent than those produced by many traditional techniques.

Modeling mosquito-borne and sexual transmission of Zika virus in an enzootic host, the African green monkey.

Mosquito-borne and sexual transmission of Zika virus (ZIKV), a TORCH pathogen, recently initiated a series of large epidemics throughout the Tropics. Animal models are necessary to determine transmission risk and study pathogenesis, as well screen antivirals and vaccine candidates. In this study, we modeled mosquito and sexual transmission of ZIKV in the African green monkey (AGM). Following subcutaneous, intravaginal or intrarectal inoculation of AGMs with ZIKV, we determined the transmission potential and infection dynamics of the virus. AGMs inoculated by all three transmission routes exhibited viremia and viral shedding followed by strong virus neutralizing antibody responses, in the absence of clinical illness. All four of the subcutaneously inoculated AGMs became infected (mean peak viremia: 2.9 log10 PFU/mL, mean duration: 4.3 days) and vRNA was detected in their oral swabs, with infectious virus being detected in a subset of these specimens. Although all four of the intravaginally inoculated AGMs developed virus neutralizing antibody responses, only three had detectable viremia (mean peak viremia: 4.0 log10 PFU/mL, mean duration: 3.0 days). These three AGMs also had vRNA and infectious virus detected in both oral and vaginal swabs. Two of the four intrarectally inoculated AGMs became infected (mean peak viremia: 3.8 log10 PFU/mL, mean duration: 3.5 days). vRNA was detected in oral swabs collected from both of these infected AGMs, and infectious virus was detected in an oral swab from one of these AGMs. Notably, vRNA and infectious virus were detected in vaginal swabs collected from the infected female AGM (peak viral load: 7.5 log10 copies/mL, peak titer: 3.8 log10 PFU/mL, range of detection: 5-21 days post infection). Abnormal clinical chemistry and hematology results were detected and acute lymphadenopathy was observed in some AGMs. Infection dynamics in all three AGM ZIKV models are similar to those reported in the majority of human ZIKV infections. Our results indicate that the AGM can be used as a surrogate to model mosquito or sexual ZIKV transmission and infection. Furthermore, our results suggest that AGMs are likely involved in the enzootic maintenance and amplification cycle of ZIKV.