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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.
Assuntos
Anticorpos Neutralizantes/imunologia , Febre Hemorrágica da Crimeia/imunologia , Sobreviventes , Sequência de Aminoácidos , Animais , Anticorpos Monoclonais/imunologia , Antígenos Virais/metabolismo , Fenômenos Biofísicos , Chlorocebus aethiops , Mapeamento de Epitopos , Epitopos/metabolismo , Feminino , Vírus da Febre Hemorrágica da Crimeia-Congo/imunologia , Febre Hemorrágica da Crimeia/prevenção & controle , Humanos , Imunoglobulina G/metabolismo , Masculino , Camundongos , Testes de Neutralização , Ligação Proteica , Engenharia de Proteínas , Proteínas Recombinantes/imunologia , Células Vero , Proteínas Virais/químicaRESUMO
BACKGROUND: The critical issues of sustained memory immunity following ebolavirus disease among long-term survivors (EVD) are still unclear. METHODS: Here, we examine virus-specific immune and inflammatory responses in 12 Sudan virus (SUDV) long-term survivors from Uganda's 2000-1 Gulu outbreak, 15 years after recovery following in vitro challenge. Total RNA from isolated SUDV-stimulated and unstimulated PBMCs 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 IFN 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.
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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 ß-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.
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Glicoproteínas/química , Glicoproteínas/genética , Vírus da Febre Hemorrágica da Crimeia-Congo/genética , Vírus da Febre Hemorrágica da Crimeia-Congo/metabolismo , Animais , Anticorpos Antivirais/imunologia , Clonagem Molecular , Cristalografia por Raios X , Modelos Animais de Doenças , Feminino , Glicoproteínas/metabolismo , Febre Hemorrágica da Crimeia/imunologia , Febre Hemorrágica da Crimeia/mortalidade , Febre Hemorrágica da Crimeia/prevenção & controle , Febre Hemorrágica da Crimeia/virologia , Humanos , Peptídeos e Proteínas de Sinalização Intercelular , Camundongos , Camundongos Knockout , Modelos Moleculares , Conformação Proteica , Fator de Transcrição STAT1/genética , Análise de Sequência de ProteínaRESUMO
To better understand humoral immunity following ebolavirus infection, a serological study of the humoral immune response against the individual viral proteins of Sudan ebolavirus (Gulu) in human survivors was performed. An enzyme-linked immunosorbent assay specific for full-length recombinant viral proteins NP, VP30, VP40, and GP1-649 (GP lacking the transmembrane domain) of Sudan ebolavirus (Gulu) was used as well as a plaque reduction neutralization test. Serum samples from human survivors, which were collected up to 10 years following recovery, were screened and analyzed. Results demonstrate that samples obtained 10 years following infection contain virus-specific antibodies that can neutralize virus. Neutralization correlates well with immunoreactivity against the viral proteins NP, VP30, and GP1-649. Sera from individuals who died or those with no documented infection but immunoreactive to ebolavirus did not neutralize. This work provides insight into the duration, profile of immunoreactivity, and neutralization capacity of the humoral immune response in ebolavirus survivors.
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Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/imunologia , Antígenos Virais/imunologia , Ebolavirus/imunologia , Doença pelo Vírus Ebola/imunologia , Animais , Anticorpos Neutralizantes/sangue , Anticorpos Antivirais/sangue , Antígenos Virais/sangue , Linhagem Celular , Chlorocebus aethiops , Células HEK293 , Doença pelo Vírus Ebola/sangue , Doença pelo Vírus Ebola/virologia , Humanos , Imunidade Humoral/imunologia , Imunoglobulina A/imunologia , Testes de Neutralização/métodos , Sudão , Sobreviventes , Células Vero , Proteínas do Envelope Viral/imunologiaRESUMO
Crimean-Congo hemorrhagic fever virus can cause lethal disease in humans yet there are no approved medical countermeasures. Viral glycoprotein GP38, unique to Nairoviridae, is a target of protective antibodies, but extensive mapping of the human antibody response to GP38 has not been previously performed. Here, we isolated 188 GP38-specific antibodies from human survivors of infection. Competition experiments showed that these antibodies bind across five distinct antigenic sites, encompassing eleven overlapping regions. Additionally, we reveal structures of GP38 bound with nine of these antibodies targeting different antigenic sites. Although GP38-specific antibodies were non-neutralizing, several antibodies were found to have protection 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 inform the development of broadly effective CCHFV antibody therapeutics.
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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.
Assuntos
Anticorpos Antivirais , Vírus da Febre Hemorrágica da Crimeia-Congo , Febre Hemorrágica da Crimeia , Humanos , Animais , Febre Hemorrágica da Crimeia/imunologia , Vírus da Febre Hemorrágica da Crimeia-Congo/imunologia , Anticorpos Antivirais/imunologia , Camundongos , Sobreviventes , Anticorpos Neutralizantes/imunologia , Feminino , Glicoproteínas/imunologia , Epitopos/imunologiaRESUMO
Oftentimes the etiological diagnostic differentiation between viral and bacterial infections is problematic, while clinical management decisions need to be made promptly upon admission. Thus, alternative rapid and sensitive diagnostic approaches need to be developed. Polymorphonuclear leukocytes (PMNs) or phagocytes act as major players in the defense response of the host during an episode of infection, and thereby undergo functional changes that differ according to the infections. PMNs functional activity can be characterized by quantification and localization of respiratory burst production and assessed by chemiluminescent (CL) byproduct reaction. We have assessed the functional states of PMNs of patients with acute infections in a luminol-amplified whole blood system using the component CL approach. In this study, blood was drawn from 69 patients with fever (>38 °C), and diagnosed as mainly viral or bacterial infections in origin. Data mining algorithms (C4.5, Support Vector Machines (SVM) and Naïve Bayes) were used to induce classification models to distinguish between clinical groups. The model with the best predictive accuracy was induced using C4.5 algorithm, resulting in 94.7% accuracy on the training set and 88.9% accuracy on the testing set. The method demonstrated a high predictive diagnostic value and may assist the clinician one day in the distinction between viral and bacterial infections and the choice of proper medication.
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Infecções Bacterianas/diagnóstico , Medições Luminescentes/métodos , Fagócitos/imunologia , Infecções por Vírus de RNA/diagnóstico , Doença Aguda , Algoritmos , Células Sanguíneas/imunologia , Humanos , Cinética , Luminol/química , Modelos Teóricos , Espécies Reativas de Oxigênio/metabolismo , SoftwareRESUMO
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.
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Glutamato Carboxipeptidase II/imunologia , Imunoconjugados/uso terapêutico , Glicoproteínas de Membrana/imunologia , Neoplasias da Próstata/diagnóstico por imagem , Neoplasias da Próstata/tratamento farmacológico , Anticorpos de Domínio Único/uso terapêutico , Animais , Antineoplásicos/uso terapêutico , Apoptose/efeitos dos fármacos , Camelus , Doxorrubicina/uso terapêutico , Liberação Controlada de Fármacos , Glutamato Carboxipeptidase II/metabolismo , Humanos , Imunoconjugados/imunologia , Masculino , Glicoproteínas de Membrana/metabolismo , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Camundongos Nus , Simulação de Acoplamento Molecular , Imagem Óptica , Neoplasias da Próstata/patologia , Anticorpos de Domínio Único/imunologia , Anticorpos de Domínio Único/metabolismo , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
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.
Assuntos
Ebolavirus , Doença pelo Vírus Ebola , Humanos , Evasão da Resposta Imune , Ligantes , Polissacarídeos , Proteínas do Envelope Viral/genéticaRESUMO
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.
Assuntos
Antígenos Virais/imunologia , Ebolavirus/imunologia , Doença pelo Vírus Ebola/imunologia , Interações Hospedeiro-Patógeno/imunologia , Imunidade , Proteínas Virais/imunologia , Adulto , Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/imunologia , Biomarcadores , Surtos de Doenças , Feminino , Doença pelo Vírus Ebola/epidemiologia , Doença pelo Vírus Ebola/metabolismo , Doença pelo Vírus Ebola/virologia , Humanos , Imunoglobulina G/sangue , Imunoglobulina G/imunologia , Memória Imunológica , Masculino , Pessoa de Meia-Idade , Testes de Neutralização , Transdução de Sinais , Sobreviventes , Linfócitos T/imunologia , Linfócitos T/metabolismo , Adulto JovemRESUMO
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.
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Doenças dos Bovinos/epidemiologia , Vírus da Febre Aftosa/isolamento & purificação , Febre Aftosa/epidemiologia , Febre Aftosa/virologia , Filogenia , Sorogrupo , Animais , Animais Selvagens/virologia , Búfalos/virologia , Bovinos , Estudos Transversais , Febre Aftosa/transmissão , Gado/virologia , Estudos Soroepidemiológicos , Uganda/epidemiologiaRESUMO
Recurrent bacterial peritonitis is a major complication in peritoneal dialysis (PD) patients, which is associated with polymorphonuclear leukocyte (PMN) functional changes and can be assessed by a chemiluminescent (CL) reaction. We applied a new approach of a dynamic component chemiluminescence sensor for the assessment of functional states of PMNs in a luminol-amplified whole-blood system. This method is based on the evaluation of CL kinetic patterns of stimulated PMNs, while the parallel measurements of intracellular and extracellular production of reactive oxygen species (ROS) from the same sample can be conducted. Blood was drawn from diabetic and nondiabetic patients during follow-up, and during peritonitis. Healthy medical personnel served as the control group. Chemiluminescence curves were recorded and presented as a sum of three biological components. CL kinetic parameters were calculated, and functional states of PMNs were assessed. Data mining algorithms were used to build decision tree models that can distinguish between different clinical groups. The induced classification models were used afterward for differentiating and classifying new blind cases and demonstrated good correlation with medical diagnosis (84.6% predictive accuracy). In conclusion, this novel method shows a high predictive diagnostic value and may assist in detection of PD-associated clinical states.
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Medições Luminescentes/métodos , Neutrófilos/fisiologia , Diálise Peritoneal/métodos , Peritonite/sangue , Diabetes Mellitus/sangue , Feminino , Humanos , Medições Luminescentes/instrumentação , Luminol/química , Masculino , Pessoa de Meia-Idade , Diálise Peritoneal/efeitos adversos , Peritonite/diagnóstico , Projetos Piloto , Espécies Reativas de Oxigênio/sangue , Explosão RespiratóriaRESUMO
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.
Assuntos
Anticorpos Monoclonais/biossíntese , Biotecnologia/métodos , Meios de Cultura/química , Hibridomas/citologia , Nanopartículas , Ondas de Rádio , Animais , Anticorpos Monoclonais/isolamento & purificação , Células CHO , Linhagem Celular Tumoral , Cricetinae , Cricetulus , Meios de Cultura/efeitos da radiação , Humanos , Hibridomas/metabolismo , Hibridomas/efeitos da radiação , ÁguaRESUMO
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.
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Proteínas Adaptadoras de Transdução de Sinal/biossíntese , Proteínas Adaptadoras de Transdução de Sinal/imunologia , Autoanticorpos/química , Neoplasias da Mama/metabolismo , Neoplasias Ovarianas/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/química , Anticorpos Monoclonais/química , Mama/metabolismo , Neoplasias da Mama/imunologia , Linhagem Celular Tumoral , Progressão da Doença , Ensaio de Imunoadsorção Enzimática/métodos , Feminino , Humanos , Imuno-Histoquímica/métodos , Neoplasias Ovarianas/imunologia , Projetos PilotoRESUMO
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.
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Proteínas Adaptadoras de Transdução de Sinal/biossíntese , Proteínas Adaptadoras de Transdução de Sinal/química , Anticorpos Monoclonais/química , Antígenos de Neoplasias/química , Autoanticorpos/química , Regulação Neoplásica da Expressão Gênica , Autoimunidade , Neoplasias da Mama , Linhagem Celular Tumoral , Membrana Celular/metabolismo , Citosol/metabolismo , Humanos , Imuno-Histoquímica/métodos , Cinética , Estrutura Terciária de ProteínaRESUMO
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 (R0, i.e., the number of secondary cases that result from an individual infection). Published R0 values were determined by systematic literature research and ranged from 0.37 to 20. R0s ≥ 4 realistically reflected the critical early outbreak phases and superspreading events. Based on the R0, the herd immunity threshold (Ic) was calculated using the equation Ic = 1 - (1/R0). The critical vaccination coverage (Vc) needed to provide herd immunity was determined by including the vaccine effectiveness (E) using the equation Vc = Ic/E. At an R0 of 4, the Ic 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.
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Doença pelo Vírus Ebola/prevenção & controle , Imunidade Coletiva , Programas de Imunização , Pandemias/prevenção & controle , Vacinação/métodos , Vacinas contra Ebola/administração & dosagem , Ebolavirus/imunologia , Pessoal de Saúde , Doença pelo Vírus Ebola/epidemiologia , HumanosRESUMO
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.
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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.
Assuntos
Anticorpos Antivirais/sangue , Cromatografia de Afinidade/instrumentação , Ebolavirus/imunologia , Doença pelo Vírus Ebola/sangue , Imunoglobulina G/sangue , Testes Imediatos , Fitas Reagentes/análise , Anticorpos Antivirais/imunologia , Cromatografia de Afinidade/economia , Ebolavirus/isolamento & purificação , Desenho de Equipamento , Doença pelo Vírus Ebola/imunologia , Doença pelo Vírus Ebola/virologia , Humanos , Imunoglobulina G/imunologia , Testes Imediatos/economia , Smartphone/economia , Smartphone/instrumentação , Fatores de TempoRESUMO
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.