High-resolution epitope mapping of commercial antibodies to ANCA antigens by yeast surface display.

Epitope mapping provides critical insight into antibody-antigen interactions. Epitope mapping of autoantibodies from patients with autoimmune diseases can help elucidate disease immunogenesis and guide the development of antigen-specific therapies. Similarly, epitope mapping of commercial antibodies targeting known autoantigens enables the use of those antibodies to test specific hypotheses. Anti-Neutrophil Cytoplasmic Autoantibody (ANCA) vasculitis results from the formation of autoantibodies to multiple autoantigens, including myeloperoxidase (MPO), proteinase-3 (PR3), plasminogen (PLG), and peroxidasin (PXDN). To perform high-resolution epitope mapping of commercial antibodies to these autoantigens, we developed a novel yeast surface display library based on a series of >5000 overlapping peptides derived from their protein sequences. Using both FACS and magnetic bead isolation of reactive yeast, we screened 19 commercially available antibodies to the ANCA autoantigens. This approach to epitope mapping resulted in highly specific, fine epitope mapping, down to single amino acid resolution in many cases. Our study also identified cross-reactivity between some commercial antibodies to MPO and PXDN, which suggests that patients with apparent autoantibodies to both proteins may be the result of cross-reactivity. Together, our data validate yeast surface display using maximally overlapping peptides as an excellent approach to linear epitope mapping.

Population dynamics of immunological synapse formation induced by bispecific T cell engagers predict clinical pharmacodynamics and treatment resistance.

Effector T cells need to form immunological synapses (IS) with recognized target cells to elicit cytolytic effects. Facilitating IS formation is the principal pharmacological action of most T cell-based cancer immunotherapies. However, the dynamics of IS formation at the cell population level, the primary driver of the pharmacodynamics of many cancer immunotherapies, remains poorly defined. Using classic immunotherapy CD3/CD19 bispecific T cell engager (BiTE) as our model system, we integrate experimental and theoretical approaches to investigate the population dynamics of IS formation and their relevance to clinical pharmacodynamics and treatment resistance. Our models produce experimentally consistent predictions when defining IS formation as a series of spatiotemporally coordinated events driven by molecular and cellular interactions. The models predict tumor-killing pharmacodynamics in patients and reveal trajectories of tumor evolution across anatomical sites under BiTE immunotherapy. Our models highlight the bone marrow as a potential sanctuary site permitting tumor evolution and antigen escape. The models also suggest that optimal dosing regimens are a function of tumor growth, CD19 expression, and patient T cell abundance, which confer adequate tumor control with reduced disease evolution. This work has implications for developing more effective T cell-based cancer immunotherapies.

Stabilizing proteins, simplified: A Rosetta-based webtool for predicting favorable mutations.

Many proteins have low thermodynamic stability, which can lead to low expression yields and limit functionality in research, industrial and clinical settings. This article introduces two, web-based tools that use the high-resolution structure of a protein along with the Rosetta molecular modeling program to predict stabilizing mutations. The protocols were recently applied to three genetically and structurally distinct proteins and successfully predicted mutations that improved thermal stability and/or protein yield. In all three cases, combining the stabilizing mutations raised the protein unfolding temperatures by more than 20°C. The first protocol evaluates point mutations and can generate a site saturation mutagenesis heatmap. The second identifies mutation clusters around user-defined positions. Both applications only require a protein structure and are particularly valuable when a deep multiple sequence alignment is not available. These tools were created to simplify protein engineering and enable research that would otherwise be infeasible due to poor expression and stability of the native molecule.

A conserved set of mutations for stabilizing soluble envelope protein dimers from dengue and Zika viruses to advance the development of subunit vaccines.

Dengue viruses (DENV serotypes 1-4) and Zika virus (ZIKV) are related flaviviruses that continue to be a public health concern, infecting hundreds of millions of people annually. The traditional live-attenuated virus vaccine approach has been challenging for the four DENV serotypes because of the need to achieve balanced replication of four independent vaccine components. Subunit vaccines represent an alternative approach that may circumvent problems inherent with live-attenuated DENV vaccines. In mature virus particles, the envelope (E) protein forms a homodimer that covers the surface of the virus and is the major target of neutralizing antibodies. Many neutralizing antibodies bind to quaternary epitopes that span across both E proteins in the homodimer. For soluble E (sE) protein to be a viable subunit vaccine, the antigens should be easy to produce and retain quaternary epitopes recognized by neutralizing antibodies. However, WT sE proteins are primarily monomeric at conditions relevant for vaccination and exhibit low expression yields. Previously, we identified amino acid mutations that stabilize the sE homodimer from DENV2 and dramatically raise expression yields. Here, we tested whether these same mutations raise the stability of sE from other DENV serotypes and ZIKV. We show that the mutations raise thermostability for sE from all the viruses, increase production yields from 4-fold to 250-fold, stabilize the homodimer, and promote binding to dimer-specific neutralizing antibodies. Our findings suggest that these sE variants could be valuable resources in the efforts to develop effective subunit vaccines for DENV serotypes 1 to 4 and ZIKV.

Designed, highly expressing, thermostable dengue virus 2 envelope protein dimers elicit quaternary epitope antibodies.

Dengue virus (DENV) is a worldwide health burden, and a safe vaccine is needed. Neutralizing antibodies bind to quaternary epitopes on DENV envelope (E) protein homodimers. However, recombinantly expressed soluble E proteins are monomers under vaccination conditions and do not present these quaternary epitopes, partly explaining their limited success as vaccine antigens. Using molecular modeling, we found DENV2 E protein mutations that induce dimerization at low concentrations (<100 pM) and enhance production yield by more than 50-fold. Cross-dimer epitope antibodies bind to the stabilized dimers, and a crystal structure resembles the wild-type (WT) E protein bound to a dimer epitope antibody. Mice immunized with the stabilized dimers developed antibodies that bind to E dimers and not monomers and elicited higher levels of DENV2-neutralizing antibodies compared to mice immunized with WT E antigen. Our findings demonstrate the feasibility of using structure-based design to produce subunit vaccines for dengue and other flaviviruses.

Dimerization of Dengue Virus E Subunits Impacts Antibody Function and Domain Focus.

Dengue virus (DENV) is responsible for the most prevalent and significant arthropod-borne viral infection of humans. The leading DENV vaccines are based on tetravalent live-attenuated virus platforms. In practice, it has been challenging to induce balanced and effective responses to each of the four DENV serotypes because of differences in the replication efficiency and immunogenicity of individual vaccine components. Unlike live vaccines, tetravalent DENV envelope (E) protein subunit vaccines are likely to stimulate balanced immune responses, because immunogenicity is replication independent. However, E protein subunit vaccines have historically performed poorly, in part because the antigens utilized were mainly monomers that did not display quaternary-structure epitopes found on E dimers and higher-order structures that form the viral envelope. In this study, we compared the immunogenicity of DENV2 E homodimers and DENV2 E monomers. The stabilized DENV2 homodimers, but not monomers, were efficiently recognized by virus-specific and flavivirus cross-reactive potently neutralizing antibodies that have been mapped to quaternary-structure epitopes displayed on the viral surface. In mice, the dimers stimulated 3-fold-higher levels of virus-specific neutralizing IgG that recognized epitopes different from those recognized by lower-level neutralizing antibodies induced by monomers. The dimer induced a stronger E domain I (EDI)- and EDII-targeted response, while the monomer antigens stimulated an EDIII epitope response and induced fusion loop epitope antibodies that are known to facilitate antibody-dependent enhancement (ADE). This study shows that DENV E subunit antigens that have been designed to mimic the structural organization of the viral surface are better vaccine antigens than E protein monomers.IMPORTANCE Dengue virus vaccine development is particularly challenging because vaccines have to provide protection against four different dengue virus stereotypes. The leading dengue virus vaccine candidates in clinical testing are all based on live-virus vaccine platforms and struggle to induce balanced immunity. Envelope subunit antigens have the potential to overcome these limitations but have historically performed poorly as vaccine antigens, because the versions tested previously were presented as monomers and not in their natural dimer configuration. This study shows that the authentic presentation of DENV2 E-based subunits has a strong impact on antibody responses, underscoring the importance of mimicking the complex protein structures that are found on DENV particle surfaces when designing subunit vaccines.

Focused dengue vaccine development: outwitting nature's design.

The four DENV serotypes are mosquito-borne pathogens that belong to the Flavivirus genus. These viruses present a major global health burden, being endemic in over 120 countries, causing ∼390 million reported infections yearly, with clinical symptoms ranging from mild fever to severe and potentially fatal hemorrhagic syndromes. Development of a safe and efficacious DENV vaccine is challenging because of the need to induce immunity against all four serotypes simultaneously, as immunity against one serotype can potentially enhance disease caused by a heterotypic secondary infection. So far, live-virus particle-based vaccine approaches struggle with inducing protective tetravalent immunity, while recombinant subunit approaches that use the envelope protein (E) as the major antigen, are gaining promise in preclinical studies. However, E-based subunits require further development and characterization to be used as effective vaccine antigens against DENV. In this review, we will address the shortcomings of recombinant E-based antigens and will discuss potential solutions to enhance E-based subunit antigen immunogenicity and vaccine efficacy.

Physiological temperatures reduce dimerization of dengue and Zika virus recombinant envelope proteins.

The spread of dengue (DENV) and Zika virus (ZIKV) is a major public health concern. The primary target of antibodies that neutralize DENV and ZIKV is the envelope (E) glycoprotein, and there is interest in using soluble recombinant E (sRecE) proteins as subunit vaccines. However, the most potent neutralizing antibodies against DENV and ZIKV recognize epitopes on the virion surface that span two or more E proteins. Therefore, to create effective DENV and ZIKV vaccines, presentation of these quaternary epitopes may be necessary. The sRecE proteins from DENV and ZIKV crystallize as native-like dimers, but studies in solution suggest that these dimers are marginally stable. To better understand the challenges associated with creating stable sRecE dimers, we characterized the thermostability of sRecE proteins from ZIKV and three DENV serotypes, DENV2-4. All four proteins irreversibly unfolded at moderate temperatures (46-53 °C). At 23 °C and low micromolar concentrations, DENV2 and ZIKV were primarily dimeric, and DENV3-4 were primarily monomeric, whereas at 37 °C, all four proteins were predominantly monomeric. We further show that the dissociation constant for DENV2 dimerization is very temperature-sensitive, ranging from <1 µm at 25 °C to 50 µm at 41 °C, due to a large exothermic enthalpy of binding of -79 kcal/mol. We also found that quaternary epitope antibody binding to DENV2-4 and ZIKV sRecE is reduced at 37 °C. Our observation of reduced sRecE dimerization at physiological temperature highlights the need for stabilizing the dimer as part of its development as a subunit vaccine.

Virus-Enabled Biosensor for Human Serum Albumin.

The label-free detection of human serum albumin (HSA) in aqueous buffer is demonstrated using a simple, monolithic, two-electrode electrochemical biosensor. In this device, both millimeter-scale electrodes are coated with a thin layer of a composite containing M13 virus particles and the electronically conductive polymer poly(3,4-ethylenedioxy thiophene) or PEDOT. These virus particles, engineered to selectively bind HSA, serve as receptors in this biosensor. The resistance component of the electrical impedance, Zre, measured between these two electrodes provides electrical transduction of HSA binding to the virus-PEDOT film. The analysis of sample volumes as small as 50 µL is made possible using a microfluidic cell. Upon exposure to HSA, virus-PEDOT films show a prompt increase in Zre within 5 s and a stable Zre signal within 15 min. HSA concentrations in the range from 100 nM to 5 µM are detectable. Sensor-to-sensor reproducibility of the HSA measurement is characterized by a coefficient-of-variance (COV) ranging from 2% to 8% across this entire concentration range. In addition, virus-PEDOT sensors successfully detected HSA in synthetic urine solutions.

Shear-stress-mediated refolding of proteins from aggregates and inclusion bodies.

Recombinant protein overexpression of large proteins in bacteria often results in insoluble and misfolded proteins directed to inclusion bodies. We report the application of shear stress in micrometer-wide, thin fluid films to refold boiled hen egg white lysozyme, recombinant hen egg white lysozyme, and recombinant caveolin-1. Furthermore, the approach allowed refolding of a much larger protein, cAMP-dependent protein kinase A (PKA). The reported methods require only minutes, which is more than 100 times faster than conventional overnight dialysis. This rapid refolding technique could significantly shorten times, lower costs, and reduce waste streams associated with protein expression for a wide range of industrial and research applications.

Measuring quality of life with the German Osteoporosis Quality of Life Questionnaire in women with osteoporosis.

OBJECTIVE: To evaluate quality of life in women suffering with osteoporosis with or without vertebral fractures for the first time with the German version of the osteoporosis quality of life questionnaire (OQLQ) and to correlate the German OQLQ with the medical outcome survey short-form health survey 36 (MOS SF-36). METHODS: In a cross-sectional study, the OQLQ and the MOS SF-36 were randomly administered to 100 postmenopausal osteoporotic women with a median age of 73.5 (quartiles 65.0; 80.0) years in order to evaluate their quality of life. RESULTS: Of the total number, 56 women had osteoporosis with at least one vertebral fracture and 44 women had osteoporosis without vertebral fracture. All items of the OQLQ as well as the MOS SF-36 showed significantly worse values for the women with vertebral fracture compared to those without vertebral fracture. Both questionnaires' domains evaluating physical wellbeing correlated strongly with each other, supporting the concept of convergent construct validity. The OQLQ domain "emotional function" showed higher correlations with different MOS SF-36 subscores than the MOS SF-36 subscore "role emotional" with the different OQLQ domains. CONCLUSION: The German version of the OQLQ was demonstrated to be feasible. Significantly, worse results in the German OQLQ for postmenopausal osteoporotic women compared to those without vertebral fracture revealed discriminant validity. The disease-targeted OQLQ seems to better reflect problems associated with low emotional wellbeing because of osteoporotic vertebral fracture.

The effect of age and gender on cytokine production by human peripheral blood mononuclear cells and markers of bone metabolism.

BACKGROUND: Aging has been associated with various alterations of immune functions, the musculoskeletal system and a decline of sex hormone levels. Estradiol has a central role in the regulation of bone turnover and also modulates the production of cytokines such as interleukin-1 and -6 and tumor necrosis factor-alpha. We therefore studied the effect of age and gender on cytokine production by mononuclear cells and markers of bone metabolism. METHODS: Peripheral blood mononuclear cells were isolated from young and elderly subjects; intracellular detection of cytokine production after stimulation with ionomycine and PMA (T cells) or LPS (monocytes) was performed by four color flow cytometry. Sex hormone levels and markers of bone metabolism were measured by RIA or ELISA: RESULTS: When we compared elderly to young women we found an increased proportion of T cells that were positive for interferon-gamma, interleukin-2, -4, -10 and -13. Also the percentage of cells producing interleukin-4 or interferon-gamma within the CD8(+) population was higher in the group of elderly women. In contrast, proportionally fewer monocytes of elderly women were positive for tumor necrosis factor-alpha or interleukin-6 than those of young women. In elderly men a higher percentage of T cells produced interleukin-2, -4 and -13. In the group of aged men we found a higher frequency of cells that produced interleukin-4 within the CD4(+) or CD8(+) population. Moreover, within monocytes of elderly men we found an increased percentage of cells positive for both interleukin-1beta and tumor necrosis factor-alpha. The data on markers of bone metabolism indicated an increase of bone turnover in old age. CONCLUSION: Our data demonstrate that aging is associated with significant alterations of bone metabolism and cytokine production by T cells and monocytes. For particular cytokines (interferon-gamma and interleukin-10 in T cells, interleukin-6 and tumor necrosis factor-alpha in monocytes) these changes are gender specific.