Can Modern Molecular Modeling Methods Help Find the Area of Potential Vulnerability of Flaviviruses?

Flaviviruses are single-stranded RNA viruses that have emerged in recent decades and infect up to 400 million people annually, causing a variety of potentially severe pathophysiological processes including hepatitis, encephalitis, hemorrhagic fever, tissues and capillaries damage. The Flaviviridae family is represented by four genera comprising 89 known virus species. There are no effective therapies available against many pathogenic flaviviruses. One of the promising strategies for flavivirus infections prevention and therapy is the use of neutralizing antibodies (NAb) that can disable the virus particles from infecting the host cells. The envelope protein (E protein) of flaviviruses is a three-domain structure that mediates the fusion of viral and host membranes delivering the infectious material. We previously developed and characterized 10H10 mAb which interacts with the E protein of the tick-borne encephalitis virus (TBEV) and many other flaviviruses' E proteins. The aim of this work was to analyze the structure of E protein binding sites recognized by the 10H10 antibody, which is reactive with different flavivirus species. Here, we present experimental data and 3D modeling indicating that the 10H10 antibody recognizes the amino acid sequence between the two cysteines C92-C116 of the fusion loop (FL) region of flaviviruses' E proteins. Overall, our results indicate that the antibody-antigen complex can form a rigid or dynamic structure that provides antibody cross reactivity and efficient interaction with the fusion loop of E protein.

Mapping Solvent Entrapment in Multiphase Systems by Electrogenerated Chemiluminescence.

The interfacial properties of multiphase systems are often difficult to quantify. We describe the observation and quantification of immiscible solvent entrapment on a carbonaceous electrode surface using microscopy-coupled electrogenerated chemiluminescence (ECL). As aqueous microdroplets suspended in 1,2-dichloroethane collide with a glassy carbon electrode surface, small volumes of the solvent become entrapped between the electrode and aqueous phase, resulting in an overestimation of the true microdroplet/electrode contact area. To quantify the contribution of solvent entrapment decreasing the microdroplet contact area, we drive an ECL reaction within the microdroplet phase using tris(bipyridine)ruthenium(II) chloride ([Ru(bpy)3]Cl2) as the ECL luminophore and sodium oxalate (Na2C2O4) as the co-reactant. Importantly, the hydrophilicity of sodium oxalate ensures that the reaction proceeds in the aqueous phase, permitting a clear contrast between the aqueous and 1,2-dichloroethane present at the electrode interface. With the contrast provided by ECL imaging, we quantify the microdroplet radius, apparent microdroplet contact area (aqueous + entrapped 1,2-dichloroethane), entrapped solvent contact area, and the number of entrapped solvent pockets per droplet. These data permit the extraction of the true microdroplet/electrode contact area for a given droplet, as well as a statistical assessment regarding the probability of solvent entrapment based on microdroplet size.

Single enzyme electroanalysis.

Traditional studies of enzymatic activity rely on the combined kinetics of millions of enzyme molecules to produce a product, an experimental approach that may wash out heterogeneities that exist between individual enzymes. Evaluating these properties on an enzyme-by-enzyme basis represents an unambiguous means of elucidating heterogeneities; however, the quantification of enzymatic activity at the single-enzyme level is fundamentally limited by the maximum catalytic rate, kcat, inherent to a given enzyme. For electrochemical methods measuring current, single enzymes must turn over greater than 107 molecules per second to produce a measurable signal on the order of 10-12 A. Enzymes with this capability are extremely rare in nature, with typical kcat values for biologically relevant enzymes falling between 1 and 10 000 s-1. Thus, clever amplification strategies are necessary to electrochemically detect the vast majority of enzymes. This review details the progress toward the electroanalytical detection and evaluation of single enzyme kinetics largely focused on the nanoimpact method, a chronoamperometric detection strategy that monitors the change in the current-time profile associated with stochastic collisions of freely diffusing entities (e.g., enzymes) onto a microelectrode or nanoelectrode surface. We discuss the experimental setups and methods developed in the last decade toward the quantification of single molecule enzymatic rates. Special emphasis is given to the limitations of measurement science in the observation of single enzyme activity and feasible methods of signal amplification with reasonable bandwidth.

Peptide Blocking CTLA-4 and B7-1 Interaction.

Discovery of the B7 family immune checkpoints such as CTLA-4 (CD152), PD-1 (CD279), as well as their ligands B7-1 (CD80), B7-2 (CD86), B7-H1 (PD-L1, CD274), and B7-DC (PD-L2, CD273), has opened new possibilities for cancer immunotherapy using monoclonal antibodies (mAb). The blockade of inhibitory receptors (CTLA-4 and PD-1) with specific mAb results in the activation of cancer patients' T lymphocytes and tumor rejection. However, the use of mAb in clinics has several limitations including side effects and cost of treatment. The development of new low-molecular compounds that block immune checkpoints' functional activity can help to overcome some of these limitations. In this paper, we describe a synthetic peptide (p344) containing 14 amino acids that specifically interact with CTLA-4 protein. A 3D computer model suggests that this peptide binds to the 99MYPPPY104 loop of CTLA-4 protein and potentially blocks the contact of CTLA-4 receptor with B7-1 ligand. Experimental data confirm the peptide-specific interaction with CTLA-4 and its ability to partially block CTLA-4/B7-1 binding. The identified synthetic peptide can be used for the development of novel immune checkpoint inhibitors that can block CTLA-4 functional activity for cancer immunotherapy.

Semaphorins 4A and 4D in chronic inflammatory diseases.

Long-term inflammatory processes directed at a particular endogenous or exogenous antigen, or sometimes of unknown etiology, form the pathogenetic basis for many debilitating conditions, such as cardiovascular, pulmonary, autoimmune, neurologic diseases, and cancer. Recent discoveries of neuroimmune semaphorins 4A and 4D (Sema4A and Sema4D, respectively) expression and function in the immune system and their key regulatory roles in fine tuning of inflammatory processes made them the molecules of interest for a potential immunotherapy. In this short review, we discuss the current knowledge in the Sema4A and Sema4D actions in chronic inflammation underlying the outlined above diseases.

The anti-lymphoma activities of anti-CD137 monoclonal antibodies are enhanced in FcγRIII(-/-) mice.

Agonistic monoclonal antibodies (mAbs) directed against the co-signaling molecule CD137 (4-1BB) elicit potent anti-tumor immunity in mice. This anti-tumor immunity has traditionally been thought to result from the ability of the Fab portion of anti-CD137 to function as an analog for CD137L. Although binding of CD137 by anti-CD137 mAbs has the potential to cross-link the Fc fragments, enabling Fc engagement of low to moderate affinity Fc gamma receptors (FcγR), the relative import of such Fc-FcγR interactions in mediating anti-CD137 associated anti-tumor immunity is unknown. We studied the ability of a rat anti-mouse CD137 mAb (2A) to mediate the anti-tumor response against the EL4E7 lymphoma in WT and FcγR(-/-) strains. 2A-treated FcRγ(-/-) mice had improved anti-tumor immunity against EL4E7, which could be completely recapitulated in FcγRIII(-/-) animals. These improved anti-tumor responses were associated with increased splenic CD8ß T cell and dendritic cell (DC) populations. Furthermore, there was an increase in the number of DCs expressing high levels of the CD40, CD80, and CD86 molecules that are associated with more effective antigen presentation. Our results demonstrate an unexpected inhibitory role for FcγRIII in the anti-tumor function of anti-CD137 and underscore the need to consider antibody isotype when engineering therapeutic mAbs.

Multiple treatment interruptions and protecting HIV-specific CD4 T cells enable durable CD8 T cell response and viral control.

Human Immunodeficiency Virus (HIV) remains a global health challenge, and novel approaches to improve HIV control are significantly important. The cell and gene therapy product AGT103-T was previously evaluated (NCT04561258) for safety, immunogenicity, and persistence in seven patients for up to 180 days post infusion. In this study, we sought to investigate the impact of AGT103-T treatment upon analytical treatment interruptions (ATIs). Six patients previously infused with AGT103-T were enrolled into an ATI study (NCT05540964), wherein they suspended their antiretroviral therapy (ART) until their viral load reached 100,000 copies/mL in two successive visits, or their CD4 count was reduced to below 300 cells/µL. During the ATI, all patients experienced viral rebound followed by a notable expansion in HIV specific immune responses. The participants demonstrated up to a five-fold increase in total CD8 counts over baseline approximately 1-2 weeks followed by the peak viremia. This coincided with a rise in HIV-specific CD8 T cells, which was attributed to the increase in antigen availability and memory recall. Thus, the protocol was amended to include a second ATI with the first ATI serving as an "auto-vaccination." Four patients participated in a second ATI. During the second ATI, the Gag-specific CD8 T cells were either maintained or rose in response to viral rebound and the peak viremia was substantially decreased. The patients reached a viral set point ranging from 7,000 copies/mL to 25,000 copies/mL. Upon resuming ART, all participants achieved viral control more rapidly than during the first ATI, with CD4 counts remaining within 10% of baseline measurements and without any serious adverse events or evidence of drug resistance. In summary, the rise in CD8 counts and the viral suppression observed in 100% of the study participants are novel observations demonstrating that AGT103-T gene therapy when combined with multiple ATIs, is a safe and effective approach for achieving viral control, with viral setpoints consistently below 25,000 copies/mL and relatively stable CD4 T cell counts. We conclude that HIV cure-oriented cell and gene therapy trials should include ATI and may benefit from designs that include multiple ATIs when induction of CD8 T cells is required to establish viral control.

Human gammadelta T lymphocytes induce robust NK cell-mediated antitumor cytotoxicity through CD137 engagement.

Natural killer (NK) cells are innate effector lymphocytes that control the growth of major histocompatibility complex class I negative tumors. We show here that γδ T lymphocytes, expanded in vitro in the presence isopentenylpyrophosphate (IPP), induce NK cell-mediated killing of tumors that are usually resistant to NK cytolysis. The induction of cytotoxicity toward these resistant tumors requires priming of NK cells by immobilized human immunoglobulin G1 and costimulation through CD137L expressed on activated γδ T lymphocytes. This costimulation increases NKG2D expression on the NK-cell surface, which is directly responsible for tumor cell lysis. Moreover, culturing peripheral blood mononuclear cells with zoledronic acid, a γδ T lymphocyte activating agent, enhances NK-cell direct cytotoxicity and antibody-dependent cellular cytotoxicity against hematopoietic and nonhematopoietic tumors. Our data reveal a novel function of human γδ T lymphocytes in the regulation of NK cell-mediated cytotoxicity and provide rationale for the use of strategies to manipulate the CD137 pathway to augment innate antitumor immunity.

CD137 promotes proliferation and survival of human B cells.

CD137 (4-1BB)-mediated costimulation plays an important role in directing the fate of Ag-stimulated T cells and NK cells, yet the role of CD137 in mediating B cell function is unknown. We found that CD137 is expressed in vitro on anti-Ig-stimulated peripheral blood B cells and in vivo on tonsillar B cells with an activated phenotype. In vitro CD137 expression is enhanced by CD40 stimulation and IFN-gamma and is inhibited by IL-4, -10, and -21. The expression of CD137 on activated human B cells is functionally relevant because engagement with its ligand at the time of activation stimulates B cell proliferation, enhances B cell survival, and induces secretion of TNF-alpha and -beta. Our study suggests that CD137 costimulation may play a role in defining the fate of Ag-stimulated human B cells.

Recent Advances in Potentiometric Biosensing.

Potentiometric biosensors are incredibly versatile tools with budding uses in industry, security, environmental safety, and human health. This mini-review on recent (2018-2020) advances in the field of potentiometric biosensors is intended to give a general overview of the main types of potentiometric biosensors for novices while still providing a brief but thorough summary of the novel advances and trends for experienced practitioners. These trends include the incorporation of nanomaterials, graphene, and novel immobilization materials, as well as a strong push towards miniaturized, flexible, and self-powered devices for in-field or at-home use.

Repertoire development and the control of cytotoxic/effector function in human gammadelta T cells.

T cells develop into two major populations distinguished by their T cell receptor (TCR) chains. Cells with the alphabeta TCR generally express CD4 or CD8 lineage markers and mostly fall into helper or cytotoxic/effector subsets. Cells expressing the alternate gammadelta TCR in humans generally do not express lineage markers, do not require MHC for antigen presentation, and recognize nonpeptidic antigens. We are interested in the dominant Vgamma2Vdelta2+ T cell subset in human peripheral blood and the control of effector function in this population. We review the literature on gammadelta T cell generation and repertoire selection, along with recent work on CD56 expression and defining a cytotoxic/effector lineage within the phosphoantigen-reactive Vgamma2Vdelta2 cells. A unique mechanism for MHC-independent repertoire selection is linked to the control of effector function that is vital to the role for gammadelta T cells in tumor surveillance. Better understanding of these mechanisms will improve our ability to exploit this population for tumor immunotherapy.

Isopentenyl pyrophosphate-activated CD56+ {gamma}{delta} T lymphocytes display potent antitumor activity toward human squamous cell carcinoma.

PURPOSE: The expression of CD56, a natural killer cell-associated molecule, on alphabeta T lymphocytes correlates with their increased antitumor effector function. CD56 is also expressed on a subset of gammadelta T cells. However, antitumor effector functions of CD56(+) gammadelta T cells are poorly characterized. EXPERIMENTAL DESIGN: To investigate the potential effector role of CD56(+) gammadelta T cells in tumor killing, we used isopentenyl pyrophosphate and interleukin-2-expanded gammadelta T cells from peripheral blood mononuclear cells of healthy donors. RESULTS: Thirty to 70% of expanded gammadelta T cells express CD56 on their surface. Interestingly, although both CD56(+) and CD56(-) gammadelta T cells express comparable levels of receptors involved in the regulation of gammadelta T-cell cytotoxicity (e.g., NKG2D and CD94), only CD56(+) gammadelta T lymphocytes are capable of killing squamous cell carcinoma and other solid tumor cell lines. This effect is likely mediated by the enhanced release of cytolytic granules because CD56(+) gammadelta T lymphocytes expressed higher levels of CD107a compared with CD56(-) controls following exposure to tumor cell lines. Lysis of tumor cell lines is blocked by concanamycin A and a combination of anti-gammadelta T-cell receptor + anti-NKG2D monoclonal antibody, suggesting that the lytic activity of CD56(+) gammadelta T cells involves the perforin-granzyme pathway and is mainly gammadelta T-cell receptor/NKG2D dependent. Importantly, CD56-expressing gammadelta T lymphocytes are resistant to Fas ligand and chemically induced apoptosis. CONCLUSIONS: Our data indicate that CD56(+) gammadelta T cells are potent antitumor effectors capable of killing squamous cell carcinoma and may play an important therapeutic role in patients with head and neck cancer and other malignancies.

Provision of antigen and CD137 signaling breaks immunological ignorance, promoting regression of poorly immunogenic tumors.

Treatment of advanced, poorly immunogenic tumors in animal models, considered the closest simulation available thus far for conditions observed in cancer patients, remains a major challenge for cancer immunotherapy. We reported previously that established tumors in mice receiving an agonistic mAb to the T cell costimulatory molecule 4-1BB (CD137) regress due to enhanced tumor antigen-specific cytotoxic T lymphocyte responses. In this study, we demonstrate that several poorly immunogenic tumors, including C3 tumor, TC-1 lung carcinoma, and B16-F10 melanoma, once established as solid tumors or metastases, are refractory to treatment by anti-4-1BB mAb. We provide evidence that immunological ignorance, rather than anergy or deletion, of tumor antigen--specific CTLs during the progressive growth of tumors prevents costimulation by anti-4-1BB mAb. Breaking CTL ignorance by immunization with a tumor antigen-derived peptide, although insufficient to stimulate a curative CTL response, is necessary for anti--4-1BB mAb to induce a CTL response leading to the regression of established tumors. Our results suggest a new approach for immunotherapy of human cancers.

Immunosignature: Serum Antibody Profiling for Cancer Diagnostics.

Biomarkers for preclinical diagnosis of cancer are valuable tools for detection of malignant tumors at early stages in groups at risk and screening healthy people, as well as monitoring disease recurrence after treatment of cancer. However the complexity of the body's response to the pathological processes makes it virtually impossible to evaluate this response to the development of the disease using a single biomarker that is present in the serum at low concentrations. An alternative approach to standard biomarker analysis is called immunosignature. Instead of going after biomarkers themselves this approach rely on the analysis of the humoral immune response to molecular changes associated with the development of pathological processes. It is known that antibodies are produced in response to proteins expressed during cancer development. Accordingly, the changes in antibody repertoire associated with tumor growth can serve as biomarkers of cancer. Immunosignature is a highly sensitive method for antibody repertoire analysis utilizing high density peptide microarrays. In the present review we discuss modern methods for antibody detection, as well as describe the principles and applications of immunosignature in research and clinical practice.

Immunoglobulin fusion proteins as a tool for evaluation of T-cell costimulatory molecules.

Costimulatory signal(s) in addition to engagement of T cell receptor is important for optimal activation of T lymphocytes. During last decade several dozens of new costimulatory molecules, including 4-1BB, CD40, OX40, CD27, and ICOS, have been identified. It is likely that in Post-Genome Era more molecules with potential costimulatory properties will be brought to our attention. We describe here a simple and reliable strategy for evaluating the functional activities of potential costimulatory molecules using soluble fusion protein between extracellular portion of costimulatory ligand and Fc portion of mouse IgG2a.

Reassessment of CD62L as a marker of pre-effector T cells in the tumor draining lymph nodes of head and neck cancer patients.

OBJECTIVES: CD62L was evaluated as a determinant of human pre-effector T cells. STUDY DESIGN AND SETTING: Phenotype and cytokine secretion profiles of CD62L cells were determined based on activation status. RESULTS: CD62L(Low) T cells demonstrated significantly higher secretion of interleukin (IL)-10 and interferon (IFN)-gamma than did CD62L(High) T cells. After activation, the majority of cells expressed high levels of the CD62L surface marker. Postactivation levels of IL-10 production remained elevated or unchanged. In a murine B16 melanoma model, freshly isolated CD62L(Low) tumor draining lymph nodes (TDLN) T cells showed increased secretion of IL-2 and IL-4 but not of IL-10 or IFN-gamma. The surface expression of CD62L and cytokine secretion patterns were maintained after activation with concomitant increases in IL-10. CONCLUSION: Our results provide evidence that CD62L(Low) T cells in TDLNs of progressively growing squamous cell carcinoma of the head and neck differ phenotypically and functionally from those of mouse origin. SIGNIFICANCE: Characterization of this human CD62L(Low) T cell population provides initial insight regarding novel surface markers in TDLN T cells that might correlate with antitumor reactivity.

BTNL8, a butyrophilin-like molecule that costimulates the primary immune response.

The role of the B7 family molecules in the regulation of the immune response is well documented. A large body of experimental evidence indicates that costimulatory molecules such as B7-1, B7-2, B7-DC, B7-H1, B7-H2, B7-H3 and B7-H4 are critical for initiation, maintenance and down-regulation of the immune response. However the immunological function of butyrophilin (BTN)-like molecules, which are a part of the expanded B7 family, is not known. Here, we demonstrate that the extracellular portion of human BTNL8 can augment Ag-induced activation of T lymphocytes. BTNL8 has two alternatively spliced forms: B7-like and BTN-like. Both isoforms of BTNL8 were expressed concurrently in various human tissues. A putative BTNL8 receptor was detected only on resting T lymphocytes. Administration of BTNL8Ig fusion protein into mice promoted production of Ag-specific IgG during the primary, but not the secondary immune responses. BTNL8 may therefore play an essential role in priming of naïve T lymphocytes.

Activation of cytokine-producing and antitumor activities of natural killer cells and macrophages by engagement of Toll-like and NOD-like receptors.

Macrophages and natural killer (NK) cells are important antitumor effectors by virtue of their ability to produce cytokines, chemokines and interferons (IFNs) and to mediate tumor cytotoxicity. Little is known about the impact of Toll-like receptor (TLR) and nucleotide binding and oligomerization domain (NOD)-like receptor (NLR) pathways on NK cell functions, and the role of TLRs and NLRs in macrophage activation is incompletely understood. In this study, we examined the capacities of expressed TLRs and NLRs to elicit cytokine production in human NK cells and THP1 macrophages, and to activate NK cytotoxicity against the squamous cell carcinoma of head and neck cell line Tu167 and erythroleukemia K562 cells. We found that NK cells express high levels of NOD2, NLRP3, TLR3, TLR7, and TLR9, while NOD1 was expressed at low levels. All tested NLR and TLR agonists potentiated NK cytotoxicity against Tu167 cells, whereas only poly (I:C) increased NK cytotoxicity against K562 cells. Poly (I:C) and Escherichia coli RNA markedly up-regulated TNF-α and IFN-γ expression in the NK92 cell line and human CD56(+)CD3(-) primary NK cells. High levels of NOD2, TLR7 and TLR9 proteins were observed in human THP1 cells, followed by TLR3, NOD1, and NLRP3. Stimulation of NLRP3 with E. coli RNA led to the highest induction of TNF-α, IL-6, IL-12p40, RANTES and IFN-ß, whereas TLR7, TLR3, TLR9, NOD1 and NOD2 agonists had lower effects. Our data reveal involvement of TLRs and NLRs in potentiation of antitumor cytotoxicity and cytokine-producing activities of human NK cells and macrophages.

Interleukin-7 inhibits tumor-induced CD27-CD28- suppressor T cells: implications for cancer immunotherapy.

PURPOSE: We have previously reported that many types of tumors can induce changes in human T cells that lead to the acquisition of suppressive function and phenotypic alterations resembling those found in senescent T cells. In the present study, we find a role for interleukin 7 (IL-7) in protecting T cells from these changes and further define involved signaling pathways. EXPERIMENTAL DESIGN: We evaluated the ability of IL-7 treatment to prevent the gain of suppressive function and phenotypic alterations in human T cells after a short coculture with tumor cells in vitro. We then used inhibitors of components of the phosphoinositide 3-kinase (PI3K)/AKT pathway and short interfering RNA knockdown of Mcl-1 and Bim to evaluate the role of these signaling pathways in IL-7 protection. RESULTS: We found that IL-7 inhibits CD27/CD28 loss and maintains proliferative capacity, IL-2 production, and reduced suppressive function. The protective ability of IL-7 depended on activation of the PI3K/AKT pathway, which inhibited activation of glycogen synthase kinase 3ß, which, in turn, prevented the phosphorylation and loss of Mcl-1. We further showed a key role for Mcl-1 in that its knockdown or inhibition abrogated the effects of IL-7. In addition, knockdown of the Mcl-1 binding partner and proapoptotic protein Bim protected T cells from these dysfunctional alterations. CONCLUSION: These observations confirm the role for Bcl-2 family members in cytokine signaling and suggest that IL-7 treatment in combination with other immunotherapies could lead to new clinical strategies to maintain normal T-cell function and reduce tumor-induced generation of dysfunctional and suppressor T cells.

Tumor-induced senescent T cells with suppressor function: a potential form of tumor immune evasion.

Senescent and suppressor T cells are reported to be increased in select patients with cancer and are poor prognostic indicators. Based on the association of these T cells and poor outcomes, we hypothesized that tumors induce senescence in T cells, which negatively effects antitumor immunity. In this report, we show that human T cells from healthy donors incubated with tumor for only 6 h at a low tumor to T-cell ratio undergo a senescence-like phenotype, characterized by the loss of CD27 and CD28 expression and telomere shortening. Tumor-induced senescence of T cells is induced by soluble factors and triggers increases in expression of senescence-associated molecules such as p53, p21, and p16. Importantly, these T cells are not only phenotypically altered, but also functionally altered as they can suppress the proliferation of responder T cells. This suppression requires cell-to-cell contact and is mediated by senescent CD4(+) and CD8(+) subpopulations, which are distinct from classically described natural T regulatory cells. Our observations support the novel concept that tumor can induce senescent T cells with suppressor function and may effect both the diagnosis and treatment of cancer.