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Abstract Background We have been studying the native autoimmune response to cancer through the isolation of human monoclonal antibodies that are cancer specific from cancer patients. To facilitate this work we previously developed a fusion partner cell line for human lymphocytes, MFP-2, that fuses efficiently with both human lymph node lymphocytes and peripheral blood lymphocytes. Using this unique trioma fusion partner cell line we isolated a panel of autologous human monoclonal antibodies, from both peripheral blood and lymph node lymphocytes, which are representative of the native repertoire of anti-cancer specific antibodies from breast cancer patients. Methods The current study employs immunocytochemistry, immunohistochemistry, Western blot analysis as well as Northern blots, Scatchard binding studies and finally SEREX analysis for target antigen identification. Results By application of an expression cloning technique known as SEREX, we determined that the target antigen for two monoclonal antibodies, 27.B1 and 27.F7, derived from lymph node B-cells of a breast cancer patient, is the PDZ domain-containing protein known as GIPC1. This protein is highly expressed not only in cultured human breast cancer cells, but also in primary and metastatic tumor tissues and its overexpression appears to be cancer cell specific. Confocal microscopy revealed cell membrane and cytoplasmic localization of the target protein, which is consistent with previous studies of this protein. Conclusion We have determined that GIPC1 is a novel breast cancer-associated immunogenic antigen that is overexpressed in breast cancer. Its role, however, in the initiation and/or progression of breast cancer remains unclear and needs further clarification.

Abstract Background We have been studying the native humoral immune response to cancer and have isolated a library of fully human autoantibodies to a variety of malignancies. We previously described the isolation and characterization of two fully human monoclonal antibodies, 27.F7 and 27.B1, from breast cancer patients that target the protein known as GIPC1, an accessory PDZ-domain binding protein involved in regulation of G-protein signaling. Human monoclonal antibodies, 27.F7 and 27.B1, to GIPC1 demonstrate specific binding to malignant breast cancer tissue with no reactivity with normal breast tissue. Methods The current study employs cELISA, flow cytometry, Western blot analysis as well as immunocytochemistry, and immunohistochemistry. Data is analyzed statistically with the Fisher one-tail and two-tail tests for two independent samples. Results By screening several other cancer cell lines with 27.F7 and 27.B1 we found consistently strong staining of other human cancer cell lines including SKOV-3 (an ovarian cancer cell line). To further clarify the association of GIPC1 with breast and ovarian cancer we carefully studied 27.F7 and 27.B1 using immunocytochemical and immunohistochemical techniques. An immunohistochemical study of normal ovarian tissue, benign, borderline and malignant ovarian serous tumors, and different types of breast cancer revealed high expression of GIPC1 protein in neoplastic cells. Interestingly, antibodies 27.F7 and 27.B1 demonstrate differential staining of borderline ovarian tumors. Examination of different types of breast cancer demonstrates that the level of GIPC1 expression depends on tumor invasiveness and displays a higher expression than in benign tumors. Conclusion The present pilot study demonstrates that the GIPC1 protein is overexpressed in ovarian and breast cancer, which may provide an important diagnostic and prognostic marker and will constitute the basis for further study of the role that this protein plays in malignant diseases. In addition, this study suggests that human monoclonal antibodies 27.F7 and 27.B1 should be further evaluated as potential diagnostic tools.

Abstract Background The isolation and production of human monoclonal antibodies is becoming an increasingly important pursuit as biopharmaceutical companies migrate their drug pipelines away from small organic molecules. As such, optimization of monoclonal antibody technologies is important, as this is becoming the new rate-limiting step for discovery and development of new pharmaceuticals. The major limitations of this system are the efficiency of isolating hybridoma clones, the process of stabilizing these clones and optimization of hybridoma cell secretion, especially for large-scale production. Many previous studies have demonstrated how perturbations in the aqueous environment can impact upon cell biology. In particular, radio frequency (RF) irradiation of solutions can have dramatic effects on behavior of solutions, cells and in particular membrane proteins, although this effect decays following removal of the RF. Recently, it was shown that nanoparticle doping of RF irradiated water (NPD water) produced a stabilized aqueous medium that maintained the characteristic properties of RF irradiated water for extended periods of time. Therefore, the ordering effect in water of the RF irradiation can now be studied in systems that required prolonged periods for analysis, such as eukaryotic cell culture. Since the formation of hybridoma cells involves the formation of a new membrane, a process that is affected by the surrounding aqueous environment, we tested these nanoparticle doped aqueous media formulations on hybridoma cell production. Results In this study, we tested the entire process of isolation and production of human monoclonal antibodies in NPD water as a means for further enhancing human monoclonal antibody isolation and production. Our results indicate an overall enhancement of hybridoma yield, viability, clonability and secretion. Furthermore, we have demonstrated that immortal cells proliferate faster whereas primary human fibroblasts proliferate slower in NPD water. Conclusion Overall, these studies indicate that NPD water can enhance cell proliferation, clonability and secretion. Furthermore, the results support the hypothesis that NPD water is effectively composed of stable microenvironments.

Background: We have been studying the native autoimmune response to cancer through the isolation of human monoclonal antibodies that are cancer specific from cancer patients. To facilitate this work we previously developed a fusion partner cell line for human lymphocytes, MFP-2, that fuses efficiently with both human lymph node lymphocytes and peripheral blood lymphocytes. Using this unique trioma fusion partner cell line we isolated a panel of autologous human monoclonal antibodies, from both peripheral blood and lymph node lymphocytes, which are representative of the native repertoire of anti-cancer specific antibodies from breast cancer patients.Methods: The current study employs immunocytochemistry, immunohistochemistry, Western blot analysis as well as Northern blots, Scatchard binding studies and finally SEREX analysis for target antigen identification.Results: By application of an expression cloning technique known as SEREX, we determined that the target antigen for two monoclonal antibodies, 27.B1 and 27.F7, derived from lymph node B-cells of a breast cancer patient, is the PDZ domain-containing protein known as GIPC1. This protein is highly expressed not only in cultured human breast cancer cells, but also in primary and metastatic tumor tissues and its overexpression appears to be cancer cell specific. Confocal microscopy revealed cell membrane and cytoplasmic localization of the target protein, which is consistent with previous studies of this protein.Conclusion: We have determined that GIPC1 is a novel breast cancer-associated immunogenic antigen that is overexpressed in breast cancer. Its role, however, in the initiation and/or progression of breast cancer remains unclear and needs further clarification. [ABSTRACT FROM AUTHOR]

The microbiome plays a vital role in human health, with changes in its composition impacting various aspects of the body. Posttranslational modification (PTM) regulates protein activity by attaching chemical groups to amino acids in an enzymatic or non-enzymatic manner. PTMs offer fast and dynamic regulation of protein expression and can be influenced by specific dietary components that induce PTM events in gut microbiomes and their hosts. PTMs on microbiome proteins have been found to contribute to host-microbe interactions. For example, in Escherichia coli , S-sulfhydration of tryptophanase regulates uremic toxin production and chronic kidney disease in mice. On a broader microbial scale, the microbiomes of patients with inflammatory bowel disease exhibit distinct PTM patterns in their metaproteomes. Moreover, pathogens and commensals can alter host PTM profiles through protein secretion and diet-regulated metabolic shifts. The emerging field of metaPTMomics focuses on understanding PTM profiles in the microbiota, their association with lifestyle factors like diet, and their functional effects on host-microbe interactions., Competing Interests: The authors declare no conflict of interest.

Background: The critical issues of sustained memory immunity following ebolavirus disease among long-term survivors are still unclear., Methods: Here, we examine virus-specific immune and inflammatory responses following in vitro challengd in 12 Sudan virus (SUDV) long-term survivors from Uganda's 2000-2001 Gulu outbreak, 15 years after recovery. Total RNA from isolated SUDV-stimulated and unstimulated peripheral blood mononuclear cells was extracted and analyzed. Matched serum samples were also collected to determine SUDV IgG levels and functionality., Results: We detected persistent humoral (58%, 7 of 12) and cellular (33%, 4 of 12) immune responses in SUDV long-term survivors and identified critical molecular mechanisms of innate and adaptive immunity. Gene expression in immune pathways, the interferon signaling system, antiviral defense response, and activation and regulation of T- and B-cell responses were observed. SUDV long-term survivors also maintained robust virus-specific IgG antibodies capable of polyfunctional responses, including neutralizing and innate Fc effector functions., Conclusions: Data integration identified significant correlations among humoral and cellular immune responses and pinpointed a specific innate and adaptive gene expression signature associated with long-lasting immunity. This could help identify natural and vaccine correlates of protection against ebolavirus disease., Competing Interests: Potential conflicts of interest. All authors: No reported conflicts. All authors have submitted the ICMJE Form for Disclosure of Potential Conflicts of Interest. Conflicts that the editors consider relevant to the content of the manuscript have been disclosed., (© The Author(s) 2023. Published by Oxford University Press on behalf of Infectious Diseases Society of America. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Crimean-Congo hemorrhagic fever virus can cause lethal disease in humans yet there are no approved medical countermeasures. Viral glycoprotein GP38, exclusive to Nairoviridae, is a target of protective antibodies and is a key antigen in preclinical vaccine candidates. Here, we isolate 188 GP38-specific antibodies from human survivors of infection. Competition experiments show that these antibodies bind across 5 distinct antigenic sites, encompassing 11 overlapping regions. Additionally, we show structures of GP38 bound with 9 of these antibodies targeting different antigenic sites. Although these GP38-specific antibodies are non-neutralizing, several display protective efficacy equal to or better than murine antibody 13G8 in two highly stringent rodent models of infection. Together, these data expand our understanding regarding this important viral protein and may inform the development of broadly effective CCHFV antibody therapeutics., Competing Interests: Declaration of interests E.C., J.L., and N.T.P. are employees and shareholders of Adimab, LLC. O.S.S., V.H., M.D., D.P.M., and L.M.W. are shareholders of Adimab., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Background: Psychosocial vulnerabilities (e.g. inadequate social support, financial insecurity, stress) and substance use elevate risks for adverse perinatal outcomes and maternal mental health morbidities. However, various barriers, including paucity of validated, simple and usable comprehensive instruments, impede execution of the recommendations to screen for such vulnerabilities in the first antenatal care visit. The current study presents findings from a newly implemented self-report tool created to overcome screening barriers in outpatient antenatal clinics., Methods: This was a retrospective chart-review of 904 women who completed the Profile for Maternal & Obstetric Treatment Effectiveness (PROMOTE) during their first antenatal visit between June and December 2019. The PROMOTE includes the 4-item NIDA Quick Screen and 15 additional items that each assess a different psychosocial vulnerability. Statistical analysis included evaluation of missing data, and exploration of missing data patterns using univariate correlations and hierarchical clustering., Results: Three quarters of women (70.0%) had no missing items. In the entire sample, all but four PROMOTE items (opioid use, planned pregnancy, educational level, and financial state) had < 5% missing values, suggesting good acceptability and feasibility. Several respondent-related characteristics such as lower education, less family support, and greater stress were associated with greater likelihood of missing items. Instrument-related characteristics associated with missing values were completing the PROMOTE in Spanish or question positioning at the end of the instrument., Conclusions and Implications: Conducting a comprehensive screening of theoretically and clinically meaningful vulnerabilities in an outpatient setting is feasible. Study findings will inform modifications of the PROMOTE and subsequent digitisation.

The intestinal epithelium plays critical roles in sensing and integrating dietary and microbial signals. How microbiota and intestinal epithelial cell (IEC) interactions regulate host physiology in the proximal small intestine, particularly the duodenum, is unclear. Using single-cell RNA sequencing of duodenal IECs under germ-free (GF) and different conventional microbiota compositions, we show that specific microbiota members alter epithelial homeostasis by increasing epithelial turnover rate, crypt proliferation, and major histocompatibility complex class II (MHCII) expression. Microbiome profiling identified Faecalibaculum rodentium as a key species involved in this regulation. F. rodentium decreases enterocyte expression of retinoic-acid-producing enzymes Adh1, Aldh1a1, and Rdh7, reducing retinoic acid signaling required to maintain certain intestinal eosinophil populations. Eosinophils suppress intraepithelial-lymphocyte-mediated production of interferon-γ that regulates epithelial cell function. Thus, we identify a retinoic acid-eosinophil-interferon-γ-dependent circuit by which the microbiota modulates duodenal epithelial homeostasis., Competing Interests: Declaration of interests W.S.G. is on the SAB of Senda Biosciences, Freya Biosciences, and Artizan Biosciences, all outside the current work., (Copyright © 2022 Elsevier Inc. All rights reserved.)

Crimean-Congo hemorrhagic fever virus (CCHFV) is a World Health Organization priority pathogen. CCHFV infections cause a highly lethal hemorrhagic fever for which specific treatments and vaccines are urgently needed. Here, we characterize the human immune response to natural CCHFV infection to identify potent neutralizing monoclonal antibodies (nAbs) targeting the viral glycoprotein. Competition experiments showed that these nAbs bind six distinct antigenic sites in the Gc subunit. These sites were further delineated through mutagenesis and mapped onto a prefusion model of Gc. Pairwise screening identified combinations of non-competing nAbs that afford synergistic neutralization. Further enhancements in neutralization breadth and potency were attained by physically linking variable domains of synergistic nAb pairs through bispecific antibody (bsAb) engineering. Although multiple nAbs protected mice from lethal CCHFV challenge in pre- or post-exposure prophylactic settings, only a single bsAb, DVD-121-801, afforded therapeutic protection. DVD-121-801 is a promising candidate suitable for clinical development as a CCHFV therapeutic., Competing Interests: Declaration of interests K.C. is a scientific advisory board member of Integrum Scientific and Biovaxys Technology Corporation. K.C., J.S.M., and J.R.L. are scientific advisory board members of the Pandemic Security Initiative of Celdara Medical. N.T.P. and L.M.W. are employees and shareholders of Adimab. D.P.M. is a shareholder of Adimab. Z.A.B., D.M.A., C.L.M., and L.Z. are shareholders and employees of Mapp Biopharmaceutical. Mapp Biopharmaceutical has filed a patent application related to this work., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Objective: Rapid, high-volume screening programs are needed as part of cervical cancer prevention in China., Methods: In a 5-day screening project in Inner Mongolia, 3345 women volunteered following a community awareness campaign, and self-swabbed to permit rapid HPV testing. Two AmpFire™ HPV detection systems (Atila Biosystems) were sufficient to provide pooled 15-HPV type data within an hour. HPV+ patients had same-day digital colposcopy (DC) performed by 1 of 6 physicians, using the EVA™ system (MobileODT). Digital images were obtained and, after biopsy of suspected lesions for later confirmatory diagnosis, women were treated immediately based on colposcopic impression. Suspected low- grade lesions were offered treatment with thermal ablation (Wisap), and suspected high-grade lesions were treated with LLETZ., Results: Of 3345 women screened, 624 (18.7%) were HPV+. Of these, 88.5% HPV+ women underwent same-day colposcopy and 78 were treated. Later consensus histology results obtained on 197 women indicated 20 CIN2+, of whom 15 were detected and treated/referred at screening (10 by thermal ablation, 4 by LLETZ, 1 by referral)., Conclusions: Global control of cervical cancer will require both vaccination and screening of a huge number of women. This study illustrates a cervical screening strategy that can be used to screen-and-treat large numbers of women. HPV self-sampling facilitates high-volume screening. Specimens can be tested rapidly, promoting minimal loss-to-follow-up. Specifically, the AmpFire™ system used in this study is highly portable, simple, rapid (92 specimens per 65 min per unit), and economical. Visual triage can be performed on HPV+ women with a portable digital colposcope that provides magnification, lighting, and a recorded image. Diagnosis and appropriate treatment remain the most subjective elements. The digital image is under study for deep-learning based automated evaluation that could assist the management decision, either by itself or combined with HPV typing., Competing Interests: Competing interestsAndrew Goldstein: President of the Gynecologic Cancers Research Foundation: a non-profit 501(c)3 corporation. Received research funding from the Gynecologic Cancers Research Foundation. Yang Lei: none to report. Lena Goldstein: none to report. Amelia Goldstein: none to report. Qiao Xu Bai: none to report. Juan Felix: none to report. Roberta Lipson: Director, United Foundation for China’s Health, a non-profit 501(c)3 corporation. Shareholder in MobileODT. Maria Demarco, Mark Schiffman, Didem Egemen, Kanan Desai: NCI has received cervical screening supplies and results at no cost from several companies (Roche, BD, Qiagen, MobileODT) for independent evaluations of test performance. Sarah Bedell: none to report. Janet Gersten: none to report. Gail Goldstein: Is a board member of the Gynecologic Cancers Research Foundation: a non-profit 501(c)3 corporation. Karen O’Keefe: none to report. Casey O’Keefe: none to report. Tierney O’Keefe: none to report. Cathy Sebag: Was an employee of MobileODT during the data collection. Lior Lobel: Was an employee of MobileODT during the data collection. Anna Zhao: employee, United Foundation for China’s Health, a non-profit 501(c)3 corporation. Yan Ling Lu: none to report., (© The Author(s) 2020.)

Associations between chronic kidney disease (CKD) and the gut microbiota have been postulated, yet questions remain about the underlying mechanisms. In humans, dietary protein increases gut bacterial production of hydrogen sulfide (H 2 S), indole, and indoxyl sulfate. The latter are uremic toxins, and H 2 S has diverse physiological functions, some of which are mediated by posttranslational modification. In a mouse model of CKD, we found that a high sulfur amino acid-containing diet resulted in posttranslationally modified microbial tryptophanase activity. This reduced uremic toxin-producing activity and ameliorated progression to CKD in the mice. Thus, diet can tune microbiota function to support healthy host physiology through posttranslational modification without altering microbial community composition., (Copyright © 2020 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)

The repertoire of methods for the detection and chemotherapeutic treatment of prostate cancer (PCa) is currently limited. Prostate-specific membrane antigen (PSMA) is overexpressed in PCa tumors and can be exploited for both imaging and drug delivery. We developed and characterized four nanobodies that present tight and specific binding and internalization into PSMA + cells and that accumulate specifically in PSMA + tumors. We then conjugated one of these nanobodies to the cytotoxic drug doxorubicin, and we show that the conjugate internalizes specifically into PSMA + cells, where the drug is released and induces cytotoxic activity. In vivo studies show that the extent of tumor growth inhibition is similar when mice are treated with commercial doxorubicin and with a 42-fold lower amount of the nanobody-conjugated doxorubicin, attesting to the efficacy of the conjugated drug. These data highlight nanobodies as promising agents for the imaging of PCa tumors and for the targeted delivery of chemotherapeutic drugs.

Crimean-Congo hemorrhagic fever virus (CCHFV) is the causative agent of the most widespread tick-borne viral infection in humans. CCHFV encodes a secreted glycoprotein (GP38) of unknown function that is the target of a protective antibody. Here, we present the crystal structure of GP38 at a resolution of 2.5 Å, which revealed a novel fold primarily consisting of a 3-helix bundle and a β-sandwich. Sequence alignment and homology modeling showed distant homology between GP38 and the ectodomain of Gn (a structural glycoprotein in CCHFV), suggestive of a gene duplication event. Analysis of convalescent-phase sera showed high titers of GP38 antibodies indicating immunogenicity in humans during natural CCHFV infection. The only protective antibody for CCHFV in an adult mouse model reported to date, 13G8, bound GP38 with subnanomolar affinity and protected against heterologous CCHFV challenge in a STAT1-knockout mouse model. Our data strongly suggest that GP38 should be evaluated as a vaccine antigen and that its structure provides a foundation to investigate functions of this protein in the viral life cycle. IMPORTANCE Crimean-Congo hemorrhagic fever virus (CCHFV) is a priority pathogen that poses a high risk to public health. Due to the high morbidity and mortality rates associated with CCHFV infection, there is an urgent need to develop medical countermeasures for disease prevention and treatment. CCHFV GP38, a secreted glycoprotein of unknown function unique to the Nairoviridae family, was recently shown to be the target of a protective antibody against CCHFV. Here, we present the crystal structure of GP38, which revealed a novel fold with distant homology to another CCHFV glycoprotein that is suggestive of a gene duplication event. We also demonstrate that antibody 13G8 protects STAT1-knockout mice against heterologous CCHFV challenge using a clinical isolate from regions where CCHFV is endemic. Collectively, these data advance our understanding of GP38 structure and antigenicity and should facilitate future studies investigating its function., (Copyright © 2020 Mishra et al.)

The Ebola Virus (EBOV) glycoprotein (GP) sterically shields cell-membrane ligands to immune receptors such as human leukocyte antigen class-1 (HLA-I) and MHC class I polypeptide-related sequence A (MICA), thus mediating immunity evasion. It was suggested that the abundant N-glycosylation of the EBOV-GP is involved in this steric shielding. We aimed to characterize (i) the GP N-glycosylation sites contributing to the shielding, and (ii) the effect of mutating these sites on immune subversion by the EBOV-GP. The two highly glycosylated domains of GP are the mucin-like domain (MLD) and the glycan cap domain (GCD) with three and six N-glycosylation sites, respectively. We mutated the N-glycosylation sites either in MLD or in GCD or in both domains. We showed that the glycosylation sites in both the MLD and GCD domains contribute to the steric shielding. This was shown for the steric shielding of either HLA-I or MICA. We then employed the fluorescence resonance energy transfer (FRET) method to measure the effect of N-glycosylation site removal on the distance in the cell membrane between the EBOV-GP and HLA-I (HLA.A * 0201 allele). We recorded high FRET values for the interaction of CFP-fused HLA.A * 0201 and YFP-fused EBOV-GP, demonstrating the very close distance (<10 nm) between these two proteins on the cell membrane of GP-expressing cells. The co-localization of HLA-I and Ebola GP was unaffected by the disruption of steric shielding, as the removal of N-glycosylation sites on Ebola GP revealed similar FRET values with HLA-I. However, these mutations directed to N-glycosylation sites had restored immune cell function otherwise impaired due to steric shielding over immune cell ligands by WT Ebola GP. Overall, we showed that the GP-mediated steric shielding aimed to impair immune function is facilitated by the N-glycans protruding from its MLD and GCD domains, but these N-glycans are not controlling the close distance between GP and its shielded proteins., (Copyright © 2020 Iraqi, Edri, Greenshpan, Kundu, Bolel, Cahana, Ottolenghi, Gazit, Lobel, Braiman and Porgador.)

Here, we report the results of a cross-sectional study designed to monitor the circulation and genetic diversity of foot and mouth disease virus (FMDV) in Uganda between 2014 and 2017. In this study, 13,614 sera and 2,068 oral-pharyngeal fluid samples were collected from cattle and analysed to determine FMDV seroprevalence, circulating serotypes and their phylogenetic relationships. Circulation of FMDV was evidenced by the detection of antibodies against non-structural proteins of FMDV or viral isolations in all districts sampled in Uganda. Sequence analysis revealed the presence of FMDV serotypes A, O, SAT 1 and SAT 2. FMDVs belonging to serotype O, isolated from 21 districts, were the most prevalent and were classified into six lineages within two East African topotypes, namely EA-1 and EA-2. Serotype A viruses belonging to the Africa G-I topotype were isolated from two districts. SAT 1 viruses grouped within topotypes I and IV and SAT 2 viruses within topotypes VII, IV and X were isolated from six and four districts respectively. Phylogenetic analysis of SAT 1 and SAT 2 sequences from cattle clustered with historical sequences from African buffalo, indicating possible interspecies transmission at the wildlife-livestock interface. In some cases, Uganda viruses also shared similarities to viral strains recovered from other regions in East Africa. This 3-year study period provides knowledge about the geographical distribution of FMDV serotypes isolated in Uganda and insights into the genetic diversity of the multiple serotypes circulating in the country. Knowledge of circulating FMDV viruses will assist in antigenic matching studies to devise improved FMDV control strategies with vaccination and vaccine strain selection for Uganda., (© 2019 Blackwell Verlag GmbH.)

Background: Profiles of immunity developed in filovirus patients and survivors have begun to shed light on antigen-specific cellular immune responses that had been previously under-studied. However, our knowledge of the breadth and length of those responses and the viral targets which mediate long-term memory immunity still lags significantly behind., Methods: We characterized antigen-specific immune responses in whole blood samples of fifteen years post-infected survivors of the Sudan virus (SUDV) outbreak in Gulu, Uganda (2000-2001). We examined T cell and IgG responses against SUDV complete antigen and four SUDV proteins; glycoprotein (GP), nucleoprotein (NP), and viral protein 30 (VP30), and 40 (VP40)., Findings: We found survivors-maintained antigen-specific CD4+ T cell memory immune responses mediated mainly by the viral protein NP. In contrast, activated CD8+ T cell responses were nearly absent in SUDV survivors, regardless of the stimulating antigen used. Analysis of anti-viral humoral immunity revealed antigen-specific IgG antibodies against SUDV and SUDV proteins. Survivor IgGs mediated live SUDV neutralization in vitro and FcγRI and FcγRIII antibody Fc-dependent responses, mainly via antibodies to the viral proteins GP and VP40., Interpretation: We highlight the key role of several proteins, i.e., GP, NP, and VP40, to act as mediators of distinctive and sustained cellular memory immune responses in long-term SUDV survivors. We suggest that the inclusion of these viral proteins in vaccine development may best mimic survivor native memory immune responses with the potential of protecting against viral infection., Funds: This research was funded by the Defense Threat Reduction Agency (CB4088) and by the National Institute Of Allergy And Infectious Diseases of the National Institutes of Health under Award Number R01AI111516. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health., (Copyright © 2019 The Authors. Published by Elsevier B.V. All rights reserved.)

Butyrate is a microbial metabolite with pleiotropic effects. Schulthess et al. (2019) report that butyrate preconditioning of macrophages enhances their anti-bacterial preparedness by inducing anti-microbial proteins that restrict bacterial growth. This study augments understanding of how microbial metabolites shape host defense., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Effective management of foot and mouth disease (FMD) requires diagnostic tests to distinguish between infected and vaccinated animals (DIVA). To address this need, several enzyme-linked immunosorbent assay (ELISA) platforms have been developed, however, these tests vary in their sensitivity and specificity and are very expensive for developing countries. Camelid-derived single-domain antibodies fragments so-called Nanobodies, have demonstrated great efficacy for the development of serological diagnostics. This study describes the development of a novel Nanobody-based FMD 3ABC competitive ELISA, for the serological detection of antibodies against FMD Non-Structural Proteins (NSP) in Uganda cattle herds. This in-house ELISA was validated using more than 600 sera from different Uganda districts, and virus serotype specificities. The evaluation of the performance of the assay demonstrated high diagnostic sensitivity and specificity of 94 % (95 % CI: 88.9-97.2), and 97.67 % (95 % CI: 94.15-99.36) respectively, as well as the capability to detect NSP-specific antibodies against multiple FMD serotype infections. In comparison with the commercial PrioCHECK FMDV NSP-FMD test, there was a strong concordance and high correlation and agreement in the performance of the two tests. This new developed Nanobody based FMD 3ABC competitive ELISA could clearly benefit routine disease diagnosis, the establishment of disease-free zones, and the improvement of FMD management and control in endemically complex environments, such as those found in Africa.

The Ebola virus (EBOV) uses evasion mechanisms that directly interfere with host T-cell antiviral responses. By steric shielding of human leukocyte antigen class-1, the Ebola glycoprotein (GP) blocks interaction with T-cell receptors (TCRs), thus rendering T cells unable to attack virus-infected cells. It is likely that this mechanism could promote increased natural killer (NK) cell activity against GP-expressing cells by preventing the engagement of NK inhibitory receptors; however, we found that primary human NK cells were less reactive to GP-expressing HEK293T cells. This was manifested as reduced cytokine secretion, a reduction in NK degranulation, and decreased lysis of GP-expressing target cells. We also demonstrated reduced recognition of GP-expressing cells by recombinant NKG2D and NKp30 receptors. In accordance, we showed a reduced monoclonal antibody-based staining of NKG2D and NKp30 ligands on GP-expressing target cells. Trypsin digestion of the membrane-associated GP led to a recovery of the recognition of membrane-associated NKG2D and NKp30 ligands. We further showed that membrane-associated GP did not shield recognition by KIR2DL receptors; in accordance, GP expression by target cells significantly perturbed signal transduction through activating, but not through inhibitory, receptors. Our results suggest a novel evasion mechanism employed by the EBOV to specifically avoid the NK cell immune response.

The recent West African Ebola virus pandemic, which affected >28,000 individuals increased interest in anti-Ebolavirus vaccination programs. Here, we systematically analyzed the requirements for a prophylactic vaccination program based on the basic reproductive number ( R 0 , i.e., the number of secondary cases that result from an individual infection). Published R 0 values were determined by systematic literature research and ranged from 0.37 to 20. R 0 s ≥ 4 realistically reflected the critical early outbreak phases and superspreading events. Based on the R 0 , the herd immunity threshold ( I c ) was calculated using the equation I c  = 1 - (1/ R 0 ). The critical vaccination coverage ( V c ) needed to provide herd immunity was determined by including the vaccine effectiveness ( E ) using the equation V c  =  I c / E . At an R 0 of 4, the I c is 75% and at an E of 90%, more than 80% of a population need to be vaccinated to establish herd immunity. Such vaccination rates are currently unrealistic because of resistance against vaccinations, financial/logistical challenges, and a lack of vaccines that provide long-term protection against all human-pathogenic Ebolaviruses. Hence, outbreak management will for the foreseeable future depend on surveillance and case isolation. Clinical vaccine candidates are only available for Ebola viruses. Their use will need to be focused on health-care workers, potentially in combination with ring vaccination approaches.

Listeria monocytogenes (Lm) is a saprophyte and intracellular pathogen. Transition to the pathogenic state relies on sensing of host-derived metabolites, yet it remains unclear how these are recognized and how they mediate virulence gene regulation. We previously found that low availability of isoleucine signals Lm to activate the virulent state. This response is dependent on CodY, a global regulator and isoleucine sensor. Isoleucine-bound CodY represses metabolic pathways including branched-chain amino acids (BCAA) biosynthesis, however under BCAA depletion, as occurs during infection, BCAA biosynthesis is upregulated and isoleucine-unbound CodY activates virulence genes. While isoleucine was revealed as an important input signal, it was not identified how internal levels are controlled during infection. Here we show that Lm regulates BCAA biosynthesis via CodY and via a riboregulator located upstream to the BCAA biosynthesis genes, named Rli60. rli60 is transcribed when BCAA levels drop, forming a ribosome-mediated attenuator that cis-regulates the downstream genes according to BCAA supply. Notably, we found that Rli60 restricts BCAA production, essentially starving Lm, a mechanism that is directly linked to virulence, as it controls the internal isoleucine pool and thereby CodY activity. This controlled BCAA auxotrophy likely evolved to enable isoleucine to serve as a host signal and virulence effector.

Ebola virus disease causes widespread and highly fatal epidemics in human populations. Today, there is still great need for point-of-care tests for diagnosis, patient management and surveillance, both during and post outbreaks. We present a point-of-care test comprising an immunochromatographic strip and a smartphone reader, which detects and semiquantifies Ebola-specific antibodies in human survivors. We developed a Sudan virus glycoprotein monoplex platform and validated it using sera from 90 human survivors and 31 local noninfected controls. The performance of the glycoprotein monoplex was 100% sensitivity and 98% specificity compared to standard whole antigen enzyme-linked immunosorbent assay (ELISA), and it was validated with freshly collected patient samples in Uganda. Moreover, we constructed a multiplex test for simultaneous detection of antibodies against three recombinant Sudan virus proteins. A pilot study comprising 15 survivors and 5 noninfected controls demonstrated sensitivity and specificity of 100% compared to standard ELISA. Finally, we developed a second multiplex subtype assay for the identification of exposure to three related EVD species: Sudan virus, Bundibugyo virus and Ebola virus (formerly Zaire) using recombinant viral glycoprotein. This multiplex test could distinguish between the host's immunity to specific viral species and identify cross-reactive immunity. These developed serological platforms consisted of capture ligands with high specificity and sensitivity, in-house developed strips and a compatible smartphone application. These platforms enabled rapid and portable testing, data storage and sharing as well as geographical tagging of the tested individuals in Uganda. This platform holds great potential as a field tool for diagnosis, vaccine development, and therapeutic evaluation.

Background: The primary objective was to determine maximum tolerated radiation dose in patients with metastatic renal cell carcinoma on pazopanib treatment., Methods: Treatment-naïve patients received pazopanib according to standard of care. Stereotactic body radiotherapy (SBRT) was delivered concurrently to the largest metastatic lesion at day 8, 10 and 12. SBRT doses were escalated in 3 dose levels (24 Gy/3, 30 Gy/3 and 36 Gy/3). Dose level was assigned using Time-to-Event Continual Reassessment Method with the target dose-limiting toxicity rate set to 0.25., Results: Thirteen patients were included. One patient experienced dose limiting toxicity (DLT) at dose level 3 (grade 4 hypoglycemia). Maximum tolerated dose was not reached with a recommended dose of 36 Gy/3 having a probability of DLT of 11%. One-year local control was 83% (95% confidence interval 61-100) and 1-year progression-free survival was 28% (95% confidence interval 1-55)., Conclusions: SBRT in combination with pazopanib is well tolerated with good local control and response rates outside the radiation field., Trial Registration: This trial was retrospectively registered on clinicaltrials.gov( NCT02334709 ) on January 6th, 2015.

Some microbial metabolites can be immunomodulatory, but there is limited understanding of how these contribute to inter-individual variation in response to infection. In a recent study in Science, Steed et al. (2017) show that the bacterial metabolite desaminotyrosine (DAT) increases type I interferon expression, resulting in an improved immune response to influenza infection., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Until recently, immune responses in filovirus survivors remained poorly understood. Early studies revealed IgM and IgG responses to infection with various filoviruses, but recent outbreaks have greatly expanded our understanding of filovirus immune responses. Immune responses in survivors of Ebola virus (EBOV) and Sudan virus (SUDV) infections have provided the most insight, with T cell responses as well as detailed antibody responses having been characterized. Immune responses to Marburg virus (MARV), however, remain almost entirely uncharacterized. We report that immune responses in MARV survivors share characteristics with EBOV and SUDV infections but have some distinct differences. MARV survivors developed multivariate CD4 + T cell responses but limited CD8 + T cell responses, more in keeping with SUDV survivors than EBOV survivors. In stark contrast to SUDV survivors, rare neutralizing antibody responses in MARV survivors diminished rapidly after the outbreak. These results warrant serious consideration for any vaccine or therapeutic that seeks to be broadly protective, as different filoviruses may require different immune responses to achieve immunity., (© 2017 Stonier et al.)

Ebolavirus is a highly lethal pathogen, causing a severe hemorrhagic disease with a high fatality rate. To better understand immune correlates of protection by virus specific IgG, we investigated the evolution of the Fcγ receptors (FcγRs)-activating capabilities of antiviral IgG in serum samples of long recovered survivors. To this end, longitudinal serum samples from survivors of Sudan ebolavirus (SUDV) infection, studied over years, were examined for the presence of Ebola-GP specific IgG subclasses, and for their binding to FcγRs. We developed a cell-based reporter system to quantitate pathogen-specific antibody binding to FcγRIIIA, FcγRIIA, FcγRIIB and FcγRI. With this system, we demonstrate that anti-GP-specific stimulation of the FcγRI reporter by survivors' sera was substantially high one year after acute infection, with a slight reduction in activity over a decade post infection. We further demonstrate that GP-specific IgG1 is by far the seroprevalent subclass that retained and even enhanced its presence in the sera, over ten years post infection; the prevalence of other GP-specific IgG subclasses was considerably reduced over time. In accordance, GP-specific FcγRI reporter response and GP-specific total IgG1 subclass correlated in the studied group of Ebola survivors. These observations are important for further informing Ebola vaccine and therapeutic development.