Kfir Oved

A novel host-proteome signature for distinguishing between acute bacterial and viral infections.

Bacterial and viral infections are often clinically indistinguishable, leading to inappropriate patient management and antibiotic misuse. Bacterial-induced host proteins such as procalcitonin, C-reactive protein (CRP), and Interleukin-6, are routinely used to support diagnosis of infection. However, their performance is negatively affected by inter-patient variability, including time from symptom onset, clinical syndrome, and pathogens. Our aim was to identify novel viral-induced host proteins that can complement bacterial-induced proteins to increase diagnostic accuracy. Initially, we conducted a bioinformatic screen to identify putative circulating host immune response proteins. The resulting 600 candidates were then quantitatively screened for diagnostic potential using blood samples from 1002 prospectively recruited patients with suspected acute infectious disease and controls with no apparent infection. For each patient, three independent physicians assigned a diagnosis based on comprehensive clinical and laboratory investigation including PCR for 21 pathogens yielding 319 bacterial, 334 viral, 112 control and 98 indeterminate diagnoses; 139 patients were excluded based on predetermined criteria. The best performing host-protein was TNF-related apoptosis-inducing ligand (TRAIL) (area under the curve [AUC] of 0.89; 95% confidence interval [CI], 0.86 to 0.91), which was consistently up-regulated in viral infected patients. We further developed a multi-protein signature using logistic-regression on half of the patients and validated it on the remaining half. The signature with the highest precision included both viral- and bacterial-induced proteins: TRAIL, Interferon gamma-induced protein-10, and CRP (AUC of 0.94; 95% CI, 0.92 to 0.96). The signature was superior to any of the individual proteins (P<0.001), as well as routinely used clinical parameters and their combinations (P<0.001). It remained robust across different physiological systems, times from symptom onset, and pathogens (AUCs 0.87-1.0). The accurate differential diagnosis provided by this novel combination of viral- and bacterial-induced proteins has the potential to improve management of patients with acute infections and reduce antibiotic misuse.

Antibody-mediated targeting of human single-chain class I MHC with covalently linked peptides induces efficient killing of tumor cells by tumor or viral-specific cytotoxic T lymphocytes

Soluble forms of human MHC class I HLA-A2 were produced in which the peptide binding groove was uniformly occupied by a single tumor or viral-derived peptides attached via a covalent flexible peptide linker to the N terminus of a single-chain β-2-microglobulin-HLA-A2 heavy chain fusion protein. A tetravalent version of this molecule with various peptides was found to be functional. It could stimulate T cells specifically as well as bind them with high avidity. The covalently linked single chain peptide-HLA-A2 construct was next fused at its C-terminal end to a scFv antibody fragment derived from the variable domains of an anti-IL-2R α subunit-specific humanized antibody, anti-Tac. The scFv–MHC fusion was thus encoded by a single gene and produced in E. coli as a single polypeptide chain. Binding studies revealed its ability to decorate Ag-positive human tumor cells with covalent peptide single-chain HLA-A2 (scHLA-A2) molecules in a manner that was entirely dependent upon the specificity of the targeting Antibody fragment. Most importantly, the covalent scHLA-A2 molecule, when bound to the target tumor cells, could induce efficient and specific HLA-A2-restricted, peptide-specific CTL-mediated lysis. These results demonstrate the ability to generate soluble, stable, and functional single-chain HLA-A2 molecules with covalently linked peptides, which when fused to targeting antibodies, potentiate CTL killing. This new approach may open the way for the development of new immunotherapeutic strategies based on antibody targeting of natural cognate MHC ligands and CTL-based cytotoxic mechanisms.

Direct visualization of the dynamics of antigen presentation in human cells infected with cytomegalovirus revealed by antibodies mimicking TCR specificity

There are no direct means to study class I MHC presentation in human normal or diseased cells. Using CMV‐infected human cells and applying novel mAb that mimic T‐cell receptor specificity directed toward the immunogenic epitope of the viral pp65 protein presented on HLA‐A2 molecules, we directly imaged the dynamics of Ag presentation in infected cells. We demonstrate that following infection large intracellular pools of HLA‐A2/pp65 complexes are localized to the Golgi. These HLA‐A2/pp65 pools account for the majority of total HLA‐A2 molecules in infected cells. Interestingly, these large pools are sequestered inside infected cells and only a small portion of them are exported to the cell surface. Virus‐induced class I MHC down‐regulation did not affect the intracellular pool of HLA‐A2/pp65 complexes. Our data also suggest that proteasome function influences the release of class I complexes to the membrane. We present herein a new and direct molecular tool to study the dynamics of viral Ag presentation that may further elucidate the balance between immune response versus viral escape.

Predicting and controlling the reactivity of immune cell populations against cancer

Heterogeneous cell populations form an interconnected network that determine their collective output. One example of such a heterogeneous immune population is tumor‐infiltrating lymphocytes (TILs), whose output can be measured in terms of its reactivity against tumors. While the degree of reactivity varies considerably between different TILs, ranging from null to a potent response, the underlying network that governs the reactivity is poorly understood. Here, we asked whether one can predict and even control this reactivity. To address this we measured the subpopulation compositions of 91 TILs surgically removed from 27 metastatic melanoma patients. Despite the large number of subpopulations compositions, we were able to computationally extract a simple set of subpopulation‐based rules that accurately predict the degree of reactivity. This raised the conjecture of whether one could control reactivity of TILs by manipulating their subpopulation composition. Remarkably, by rationally enriching and depleting selected subsets of subpopulations, we were able to restore anti‐tumor reactivity to nonreactive TILs. Altogether, this work describes a general framework for predicting and controlling the output of a cell mixture.

Antigen-Dependent Integration of Opposing Proximal TCR-Signaling Cascades Determines the Functional Fate of T Lymphocytes

T cell anergy is a key tolerance mechanism to mitigate unwanted T cell activation against self by rendering lymphocytes functionally inactive following Ag encounter. Ag plays an important role in anergy induction where high supraoptimal doses lead to the unresponsive phenotype. How T cells “measure” Ag dose and how this determines functional output to a given antigenic dose remain unclear. Using multiparametric phospho-flow and mass cytometry, we measured the intracellular phosphorylation-dependent signaling events at a single-cell resolution and studied the phosphorylation levels of key proximal human TCR activation- and inhibition-signaling molecules. We show that the intracellular balance and signal integration between these opposing signaling cascades serve as the molecular switch gauging Ag dose. An Ag density of 100 peptide–MHC complexes/cell was found to be the transition point between dominant activation and inhibition cascades, whereas higher Ag doses induced an anergic functional state. Finally, the neutralization of key inhibitory molecules reversed T cell unresponsiveness and enabled maximal T cell functions, even in the presence of very high Ag doses. This mechanism permits T cells to make integrated “measurements” of Ag dose that determine subsequent functional outcomes.

A Novel Postpriming Regulatory Check Point of Effector/Memory T Cells Dictated through Antigen Density Threshold-Dependent Anergy

CTLs act as the effector arm of the cell-mediated immune system to kill undesirable cells. Two processes regulate these effector cells to prevent self reactivity: a thymic selection process that eliminates autoreactive clones and a multistage activation or priming process that endows them with a license to kill cognate target cells. Hitherto no subsequent regulatory restrictions have been ascribed for properly primed and activated CTLs that are licensed to kill. In this study we show that CTLs possess a novel postpriming regulatory mechanism(s) that influences the outcome of their encounter with cognate target cells. This mechanism gauges the degree of Ag density, whereupon reaching a certain threshold significant changes occur that induce anergy in the effector T cells. The biological consequences of this Ag-induced postpriming control includes alterations in the expression of cell surface molecules that control immunological synapse activity and cytokine profiles and induce retarded cell proliferation. Most profound is genome-wide microarray analysis that demonstrates changes in the expression of genes related to membrane potential, TCR signal transduction, energy metabolism, and cell cycle control. Thus, a discernible and unique gene expression signature for anergy as a response to high Ag density has been observed. Consequently, activated T cells possess properties of a self-referential sensory organ. These studies identify a new postpriming control mechanism of CTL with anergenic-like properties. This mechanism extends our understanding of the control of immune function and regulation such as peripheral tolerance, viral infections, antitumor immune responses, hypersensitivity, and autoimmunity.

Selective antibody-mediated targeting of class I MHC to EGFR-expressing tumor cells induces potent antitumor CTL activity in vitro and in vivo

Epidermal growth factor receptor (EGFR) is highly overexpressed in many tumor types. We present a new fusion molecule that can target solid tumors that express EGFR. The fusion molecule combines the advantage(s) of the well‐established tumor targeting capabilities of high affinity recombinant fragments of antibodies with the known efficient, specific and potent killing ability of CD8 T lymphocytes directed against highly antigenic MHC/peptide complexes. A recombinant chimeric molecule was created by the genetic fusion of the scFv antibody fragment derived from the anti‐EGFR monoclonal antibody C225, to monomeric single‐chain HLA‐A2 complexes containing immunodominant tumor or viral‐specific peptides. The fusion protein can induce very efficiently CTL‐dependent lysis of EGFR‐expressing tumor cells regardless of the expression of self peptide‐MHC complexes. Moreover, the molecule exhibited very potent antitumor activity in vivo in nude mice bearing preestablished human tumor xenografts. These in vitro and in vivo results indicate that recombinant scFv‐MHC‐peptide fusion molecules might represent a novel and powerful approach to immunotherapy of solid tumors, bridging antibody and T lymphocyte attack on cancer cells.

Validation of a Novel Assay to Distinguish Bacterial and Viral Infections

A novel 3-protein host-assay’s diagnostic performance for distinguishing between bacterial and viral etiologies is validated in a double-blind, investigator-driven study in febrile children. BACKGROUND: Reliably distinguishing bacterial from viral infections is often challenging, leading to antibiotic misuse. A novel assay that integrates measurements of blood-borne host-proteins (tumor necrosis factor-related apoptosis-inducing ligand, interferon γ-induced protein-10, and C-reactive protein [CRP]) was developed to assist in differentiation between bacterial and viral disease. METHODS: We performed double-blind, multicenter assay evaluation using serum remnants collected at 5 pediatric emergency departments and 2 wards from children ≥3 months to ≤18 years without (n = 68) and with (n = 529) suspicion of acute infection. Infectious cohort inclusion criteria were fever ≥38°C and symptom duration ≤7 days. The reference standard diagnosis was based on predetermined criteria plus adjudication by experts blinded to assay results. Assay performers were blinded to the reference standard. Assay cutoffs were predefined. RESULTS: Of 529 potentially eligible patients with suspected acute infection, 100 did not fulfill infectious inclusion criteria and 68 had insufficient serum. The resulting cohort included 361 patients, with 239 viral, 68 bacterial, and 54 indeterminate reference standard diagnoses. The assay distinguished between bacterial and viral patients with 93.8% sensitivity (95% confidence interval: 87.8%–99.8%) and 89.8% specificity (85.6%–94.0%); 11.7% had an equivocal assay outcome. The assay outperformed CRP (cutoff 40 mg/L; sensitivity 88.2% [80.4%–96.1%], specificity 73.2% [67.6%–78.9%]) and procalcitonin testing (cutoff 0.5 ng/mL; sensitivity 63.1% [51.0%–75.1%], specificity 82.3% [77.1%–87.5%]). CONCLUSIONS: Double-blinded evaluation confirmed high assay performance in febrile children. Assay was significantly more accurate than CRP, procalcitonin, and routine laboratory parameters. Additional studies are warranted to support its potential to improve antimicrobial treatment decisions.

A novel host-protein assay outperforms routine parameters for distinguishing between bacterial and viral lower respiratory tract infections

Bacterial and viral lower respiratory tract infections (LRTIs) are often clinically indistinguishable, leading to antibiotic overuse. We compared the diagnostic accuracy of a new assay that combines 3 host-biomarkers (TRAIL, IP-10, CRP) with parameters in routine use to distinguish bacterial from viral LRTIs. Study cohort included 184 potentially eligible pediatric and adult patients. Reference standard diagnosis was based on adjudication by an expert panel following comprehensive clinical and laboratory investigation (including respiratory PCRs). Experts were blinded to assay results and assay performers were blinded to reference standard outcomes. Evaluated cohort included 88 bacterial and 36 viral patients (23 did not fulfill inclusion criteria; 37 had indeterminate reference standard outcome). Assay distinguished bacterial from viral LRTI patients with sensitivity of 0.93±0.06 and specificity of 0.91±0.09, outperforming routine parameters, including WBC, CRP and chest x-ray signs. These findings support the assays potential to help clinicians avoid missing bacterial LRTIs or overusing antibiotics.

Automating a new host-protein assay for differentiating bacterial from viral infection to reduce operator hands-on time

Distinguishing bacterial from viral infections is often challenging, leading to antibiotic misuse, and detrimental ramifications for the patient, the healthcare system and society. A novel ELISA-based assay that integrates the circulating levels of three host-response proteins (TRAIL, IP-10 and CRP) was developed to assist in differentiation between bacterial and viral etiologies. We developed a new protocol for measuring the host-based assay biomarkers using an automated ELISA workstation. The automated protocol was validated and was able to reduce technician hands-on time by 76%, while maintaining high analytical performance. Following automation, the assay has been incorporated into the routine workflow at a pediatric department, and is performed daily on admitted and emergency department patients. The automation protocol reduces the overall burden on the hospital laboratory performing the assay. This benefit has potential to promote adoption of the host-based assay, facilitating timely triage of febrile patients and prudent use of antibiotics.