HIV-1 Activates T Cell Signaling Independently of Antigen to Drive Viral Spread.

PubMed

Len, Alice C L; Starling, Shimona; Shivkumar, Maitreyi; Jolly, Clare

2017-01-24

HIV-1 spreads between CD4 T cells most efficiently through virus-induced cell-cell contacts. To test whether this process potentiates viral spread by activating signaling pathways, we developed an approach to analyze the phosphoproteome in infected and uninfected mixed-population T cells using differential metabolic labeling and mass spectrometry. We discovered HIV-1-induced activation of signaling networks during viral spread encompassing over 200 cellular proteins. Strikingly, pathways downstream of the T cell receptor were the most significantly activated, despite the absence of canonical antigen-dependent stimulation. The importance of this pathway was demonstrated by the depletion of proteins, and we show that HIV-1 Env-mediated cell-cell contact, the T cell receptor, and the Src kinase Lck were essential for signaling-dependent enhancement of viral dissemination. This study demonstrates that manipulation of signaling at immune cell contacts by HIV-1 is essential for promoting virus replication and defines a paradigm for antigen-independent T cell signaling. Copyright © 2017 The Author(s). Published by Elsevier Inc. All rights reserved.

Multiplexed screening of natural humoral immunity identifies antibodies at fine specificity for complex and dynamic viral targets.

PubMed

McCutcheon, Krista M; Gray, Julia; Chen, Natalie Y; Liu, Keyi; Park, Minha; Ellsworth, Stote; Tripp, Ralph A; Tompkins, S Mark; Johnson, Scott K; Samet, Shelly; Pereira, Lenore; Kauvar, Lawrence M

2014-01-01

Viral entry targets with therapeutic neutralizing potential are subject to multiple escape mechanisms, including antigenic drift, immune dominance of functionally irrelevant epitopes, and subtle variations in host cell mechanisms. A surprising finding of recent years is that potent neutralizing antibodies to viral epitopes independent of strain exist, but are poorly represented across the diverse human population. Identifying these antibodies and understanding the biology mediating the specific immune response is thus difficult. An effective strategy for meeting this challenge is to incorporate multiplexed antigen screening into a high throughput survey of the memory B cell repertoire from immune individuals. We used this approach to discover suites of cross-clade antibodies directed to conformational epitopes in the stalk region of the influenza A hemagglutinin (HA) protein and to select high-affinity anti-peptide antibodies to the glycoprotein B (gB) of human cytomegalovirus. In each case, our screens revealed a restricted VH and VL germline usage, including published and previously unidentified gene families. The in vivo evolution of paratope specificity with optimal neutralizing activity was understandable after correlating biological activities with kinetic binding and epitope recognition. Iterative feedback between antigen probe design based on structure and function information with high throughput multiplexed screening demonstrated a generally applicable strategy for efficient identification of safe, native, finely tuned antibodies with the potential for high genetic barriers to viral escape.

Differential T cell response against BK virus regulatory and structural antigens: A viral dynamics modelling approach.

PubMed

Blazquez-Navarro, Arturo; Schachtner, Thomas; Stervbo, Ulrik; Sefrin, Anett; Stein, Maik; Westhoff, Timm H; Reinke, Petra; Klipp, Edda; Babel, Nina; Neumann, Avidan U; Or-Guil, Michal

2018-05-01

BK virus (BKV) associated nephropathy affects 1-10% of kidney transplant recipients, leading to graft failure in about 50% of cases. Immune responses against different BKV antigens have been shown to have a prognostic value for disease development. Data currently suggest that the structural antigens and regulatory antigens of BKV might each trigger a different mode of action of the immune response. To study the influence of different modes of action of the cellular immune response on BKV clearance dynamics, we have analysed the kinetics of BKV plasma load and anti-BKV T cell response (Elispot) in six patients with BKV associated nephropathy using ODE modelling. The results show that only a small number of hypotheses on the mode of action are compatible with the empirical data. The hypothesis with the highest empirical support is that structural antigens trigger blocking of virus production from infected cells, whereas regulatory antigens trigger an acceleration of death of infected cells. These differential modes of action could be important for our understanding of BKV resolution, as according to the hypothesis, only regulatory antigens would trigger a fast and continuous clearance of the viral load. Other hypotheses showed a lower degree of empirical support, but could potentially explain the clearing mechanisms of individual patients. Our results highlight the heterogeneity of the dynamics, including the delay between immune response against structural versus regulatory antigens, and its relevance for BKV clearance. Our modelling approach is the first that studies the process of BKV clearance by bringing together viral and immune kinetics and can provide a framework for personalised hypotheses generation on the interrelations between cellular immunity and viral dynamics.

Simultaneous Quantification of Viral Antigen Expression Kinetics Using Data-Independent (DIA) Mass Spectrometry*

PubMed Central

Croft, Nathan P.; de Verteuil, Danielle A.; Smith, Stewart A.; Wong, Yik Chun; Schittenhelm, Ralf B.; Tscharke, David C.; Purcell, Anthony W.

2015-01-01

The generation of antigen-specific reagents is a significant bottleneck in the study of complex pathogens that express many hundreds to thousands of different proteins or to emerging or new strains of viruses that display potential pandemic qualities and therefore require rapid investigation. In these instances the development of antibodies for example can be prohibitively expensive to cover the full pathogen proteome, or the lead time may be unacceptably long in urgent cases where new highly pathogenic viral strains may emerge. Because genomic information on such pathogens can be rapidly acquired this opens up avenues using mass spectrometric approaches to study pathogen antigen expression, host responses and for screening the utility of therapeutics. In particular, data-independent acquisition (DIA) modalities on high-resolution mass spectrometers generate spectral information on all components of a complex sample providing depth of coverage hitherto only seen in genomic deep sequencing. The spectral information generated by DIA can be iteratively interrogated for potentially any protein of interest providing both evidence of protein expression and quantitation. Here we apply a solely DIA mass spectrometry based methodology to profile the viral antigen expression in cells infected with vaccinia virus up to 9 h post infection without the need for antigen specific antibodies or other reagents. We demonstrate deep coverage of the vaccinia virus proteome using a SWATH-MS acquisition approach, extracting quantitative kinetics of 100 virus proteins within a single experiment. The results highlight the complexity of vaccinia protein expression, complementing what is known at the transcriptomic level, and provide a valuable resource and technique for future studies of viral infection and replication kinetics. Furthermore, they highlight the utility of DIA and mass spectrometry in the dissection of host-pathogen interactions. PMID:25755296

Viral Inhibition of the Transporter Associated with Antigen Processing (TAP): A Striking Example of Functional Convergent Evolution

PubMed Central

Verweij, Marieke C.; Horst, Daniëlle; Griffin, Bryan D.; Luteijn, Rutger D.; Davison, Andrew J.; Ressing, Maaike E.; Wiertz, Emmanuel J. H. J.

2015-01-01

Herpesviruses are large DNA viruses that are highly abundant within their host populations. Even in the presence of a healthy immune system, these viruses manage to cause lifelong infections. This persistence is partially mediated by the virus entering latency, a phase of infection characterized by limited viral protein expression. Moreover, herpesviruses have devoted a significant part of their coding capacity to immune evasion strategies. It is believed that the close coexistence of herpesviruses and their hosts has resulted in the evolution of viral proteins that specifically attack multiple arms of the host immune system. Cytotoxic T lymphocytes (CTLs) play an important role in antiviral immunity. CTLs recognize their target through viral peptides presented in the context of MHC molecules at the cell surface. Every herpesvirus studied to date encodes multiple immune evasion molecules that effectively interfere with specific steps of the MHC class I antigen presentation pathway. The transporter associated with antigen processing (TAP) plays a key role in the loading of viral peptides onto MHC class I molecules. This is reflected by the numerous ways herpesviruses have developed to block TAP function. In this review, we describe the characteristics and mechanisms of action of all known virus-encoded TAP inhibitors. Orthologs of these proteins encoded by related viruses are identified, and the conservation of TAP inhibition is discussed. A phylogenetic analysis of members of the family Herpesviridae is included to study the origin of these molecules. In addition, we discuss the characteristics of the first TAP inhibitor identified outside the herpesvirus family, namely, in cowpox virus. The strategies of TAP inhibition employed by viruses are very distinct and are likely to have been acquired independently during evolution. These findings and the recent discovery of a non-herpesvirus TAP inhibitor represent a striking example of functional convergent evolution

Identification, using sera from exposed animals, of putative viral antigens in livers of primates with callitrichid hepatitis.

PubMed Central

Stephensen, C B; Montali, R J; Ramsay, E C; Holmes, K V

1990-01-01

Callitrichid hepatitis (CH) is an acute, frequently fatal viral hepatitis which affects members of the primate family Callitrichidae (R. J. Montali, E. C. Ramsay, C. B. Stephensen, M. Worley, J. A. Davis, and K. V. Holmes, J. Infect. Dis. 160:759-765, 1989; E. C. Ramsay, R. J. Montali, M. Worley, C. B. Stephensen, and K. V. Holmes, J. Zoo Wildlife Med. 20:178-183, 1989). Outbreaks of the disease occur in zoos and animal parks. In this study, CH-specific antigens were identified in the livers of infected animals by using immune sera from primates with CH and CH-exposed asymptomatic animals. Three CH-specific antigens with apparent molecular masses of 34, 54, and 65 kDa were identified. A polyclonal antiserum was raised against the 54-kDa antigen. These antigens were not found in the livers of uninfected animals and may be viral proteins. Our results suggest that at least five of the six outbreaks of CH considered here were caused by the same virus or by an antigenically related virus. Images PMID:2123012

Strategies for targeting primate neural circuits with viral vectors

PubMed Central

El-Shamayleh, Yasmine; Ni, Amy M.

2016-01-01

Understanding how the brain works requires understanding how different types of neurons contribute to circuit function and organism behavior. Progress on this front has been accelerated by optogenetics and chemogenetics, which provide an unprecedented level of control over distinct neuronal types in small animals. In primates, however, targeting specific types of neurons with these tools remains challenging. In this review, we discuss existing and emerging strategies for directing genetic manipulations to targeted neurons in the adult primate central nervous system. We review the literature on viral vectors for gene delivery to neurons, focusing on adeno-associated viral vectors and lentiviral vectors, their tropism for different cell types, and prospects for new variants with improved efficacy and selectivity. We discuss two projection targeting approaches for probing neural circuits: anterograde projection targeting and retrograde transport of viral vectors. We conclude with an analysis of cell type-specific promoters and other nucleotide sequences that can be used in viral vectors to target neuronal types at the transcriptional level. PMID:27052579

[Immunotherapy for refractory viral infections].

PubMed

Morio, Tomohiro; Fujita, Yuriko; Takahashi, Satoshi

Various antiviral agents have been developed, which are sometimes associated with toxicity, development of virus-resistant strain, and high cost. Virus-specific T-cell (VST) therapy provides an alternative curative therapy that can be effective for a prolonged time without eliciting drug resistance. VSTs can be directly separated using several types of capture devices and can be obtained by stimulating peripheral blood mononuclear cells with viral antigens (virus, protein, or peptide) loaded on antigen-presenting cells (APC). APC can be transduced with virus-antigen coding plasmid or pulsed with overlapping peptides. VST therapy has been studied in drug non-responsive viral infections after hematopoietic cell transplantation (HCT). Several previous studies have demonstrated the efficacy of VST therapy without significant severe GVHD. In addition, VSTs from a third-party donor have been prepared and administered for post-HCT viral infection. Although target viruses of VSTs include herpes virus species and polyomavirus species, a wide variety of pathogens, such as papillomavirus, intracellular bacteria, and fungi, can be treated by pathogen-specific T-cells. Perhaps, these specific T-cells could be used for opportunistic infections in other immunocompromised hosts in the near future.

Viral proteases: an emerging therapeutic target.

PubMed

Korant, B D

1988-01-01

Only a few viral diseases are presently treatable because of our limited knowledge of specific viral target molecules. An attractive class of viral molecules toward which chemotherapeutic agents could be aimed are proteases coded by some virus groups such as retro- or picornaviruses (poliomyelitis, common cold virus). The picornavirus enzymes were discovered first, and they have now been characterized by a combination of molecular-genetic and biochemical approaches. Several laboratories have expressed the picornaviral enzymes in heterologous systems and have reported proteolytic activity, as well as the high cleavage fidelity diagnostic of the viral proteases. After dealing with several technical difficulties often encountered in standard genetic engineering approaches, one viral protease is now available to us in quantity and is amendable to mutagenic procedures. The initial outcome of the mutagenesis studies has been the confirmation of our earlier work with inhibitors, which suggested a cysteine active-site class. There is a clustering of active-site residues which may be unique to these viruses. The requirement for an active-site cysteine-histidine pair in combination with detailed information on the viral cleavage sites has permitted design of selective inhibitors with attractive antiviral properties. Future goals include investigation of the structural basis for selective processing and application of the cleavage specificity to general problems in genetic engineering.

Radioimmunoassays of hidden viral antigens

DOE Office of Scientific and Technical Information (OSTI.GOV)

Neurath, A.R.; Strick, N.; Baker, L.

1982-07-01

Antigens corresponding to infectious agents may be present in biological specimens only in a cryptic form bound to antibodies and, thus, may elude detection. We describe a solid-phase technique for separation of antigens from antibodies. Immune complexes are precipitated from serum by polyethylene glycol, dissociated with NaSCN, and adsorbed onto nitrocellulose or polystyrene supports. Antigens remain topographically separated from antibodies after removal of NaSCN and can be detected with radiolabeled antibodies. Genomes from viruses immobilized on nitrocellulose can be identified by nucleic acid hybridization. Nanogram quantities of sequestered hepatitis B surface and core antigens and picogram amounts of hepatitis Bmore » virus DNA were detected. Antibody-bound adenovirus, herpesvirus, and measles virus antigens were discerned by the procedure.« less

The search for new antigenic targets in myasthenia gravis.

PubMed

Cossins, Judith; Belaya, Katsiaryna; Zoltowska, Katarzyna; Koneczny, Inga; Maxwell, Susan; Jacobson, Leslie; Leite, Maria Isabel; Waters, Patrick; Vincent, Angela; Beeson, David

2012-12-01

Around 80% of myasthenia gravis patients have antibodies against the acetylcholine receptor, and 0-60% of the remaining patients have antibodies against the muscle-specific tyrosine kinase, MuSK. Another recently identified antigen is low-density lipoprotein receptor-related protein 4 (Lrp4). To improve the existing assays and widen the search for new antigenic targets, we have employed cell-based assays in which candidate target proteins are expressed on the cell surface of transfected cells and probed with patient sera. These assays, combined with use of myotube cultures to explore the effects of the antibodies, enable us to begin to identify new antigenic targets and test antibody pathogenicity in vitro. © 2012 New York Academy of Sciences.

Antigen-specific T cell therapies for cancer

PubMed Central

Manzo, Teresa; Heslop, Helen E.; Rooney, Cliona M.

2015-01-01

Adoptively transferred antigen-specific T cells that recognize tumor antigens through their native receptors have many potential benefits as treatment for virus-associated diseases and malignancies, due to their ability to selectively recognize tumor antigens, expand and persist to provide long-term protection. Infusions of T cells targeting Epstein–Barr virus (EBV) antigens have shown encouraging response rates in patients with post-transplant lymphoproliferative disease as well as EBV-positive lymphomas and nasopharyngeal cancer, although a recent study also showed that human papilloma virus-reactive T cells can induce complete regression of metastatic cervical cancer. This strategy is also being evaluated to target non-viral tumor-associated antigens. Targeting these less immunogenic antigens is more challenging, as tumor antigens are generally weak, and high avidity T cells specific for self-antigens are deleted in the thymus, but tumor responses have been reported. Current research focusses on defining factors that promote in vivo persistence of transferred cells and ameliorate the immunosuppressive microenvironment. To this end, investigators are evaluating the effects of combining adoptive transfer of antigen-specific T cells with other immunotherapy moieties such as checkpoint inhibitors. Genetic modification of infused T cells may also be used to overcome tumor evasion mechanisms, and vaccines may be used to promote in vivo proliferation. PMID:26160910

Equine IgE responses to non-viral vaccine components.

PubMed

Gershwin, Laurel J; Netherwood, Kristina A; Norris, Meredith Somerville; Behrens, Nicole E; Shao, Matt X

2012-12-14

Vaccination of horses is performed annually or semi-annually with multiple viral antigens, either in a combination vaccine or as separate injections. While this practice undoubtedly prevents infection from such diseases as rabies, equine influenza, West Nile virus, and equine herpes virus, the procedure is not without repercussions. Hypersensitivity reactions, including fatal anaphylactic shock, after vaccination, although uncommon, have increased in incidence in recent years. Studies reported herein document the development of IgE antibodies against non-target antigen components of equine viral vaccines. We hypothesize that viral vaccines can induce an IgE response to non-target antigens, which could elicit an adverse response after vaccination with another viral vaccine containing the same component. In one study IgE responses to components of West Nile virus vaccine were evaluated by ELISA before and after vaccination in 30 horses. In a second five-year study 77 horses were similarly tested for IgE antibodies against bovine serum albumin (BSA), a component of most viral vaccines. Mast cell sensitization was evaluated in horses with high, moderate, and negative serum BSA specific IgE using an intradermal skin test with BSA. Over the five-year period high IgE responder horses showed gradually increasing BSA specific serum IgE levels and positive skin test reactivity, yet none had an adverse event. Sera from horses that had developed adverse vaccine reactions were also tested for IgE antibodies. Several of these horses had extremely high levels of BSA-specific IgE. These data suggest that non-essential protein components of vaccines may sensitize horses for future adverse responses to vaccination. Copyright © 2012 Elsevier Ltd. All rights reserved.

Suppression of Antigen-Specific T Cell Responses by the Kaposi's Sarcoma-Associated Herpesvirus Viral OX2 Protein and Its Cellular Orthologue, CD200

PubMed Central

Misstear, Karen; Chanas, Simon A.; Rezaee, S. A. Rahim; Colman, Rachel; Quinn, Laura L.; Long, Heather M.; Goodyear, Oliver; Lord, Janet M.; Hislop, Andrew D.

2012-01-01

Regulating appropriate activation of the immune response in the healthy host despite continual immune surveillance dictates that immune responses must be either self-limiting and therefore negatively regulated following their activation or prevented from developing inappropriately. In the case of antigen-specific T cells, their response is attenuated by several mechanisms, including ligation of CTLA-4 and PD-1. Through the study of the viral OX2 (vOX2) immunoregulator encoded by Kaposi's sarcoma-associated herpesvirus (KSHV), we have identified a T cell-attenuating role both for this protein and for CD200, a cellular orthologue of the viral vOX2 protein. In vitro, antigen-presenting cells (APC) expressing either native vOX2 or CD200 suppressed two functions of cognate antigen-specific T cell clones: gamma interferon (IFN-γ) production and mobilization of CD107a, a cytolytic granule component and measure of target cell killing ability. Mechanistically, vOX2 and CD200 expression on APC suppressed the phosphorylation of ERK1/2 mitogen-activated protein kinase in responding T cells. These data provide the first evidence for a role of both KSHV vOX2 and cellular CD200 in the negative regulation of antigen-specific T cell responses. They suggest that KSHV has evolved to harness the host CD200-based mechanism of attenuation of T cell responses to facilitate virus persistence and dissemination within the infected individual. Moreover, our studies define a new paradigm in immune modulation by viruses: the provision of a negative costimulatory signal to T cells by a virus-encoded orthologue of CD200. PMID:22491458

Tumor-targeting domains for chimeric antigen receptor T cells.

PubMed

Bezverbnaya, Ksenia; Mathews, Ashish; Sidhu, Jesse; Helsen, Christopher W; Bramson, Jonathan L

2017-01-01

Immunotherapy with chimeric antigen receptor (CAR) T cells has been advancing steadily in clinical trials. Since the ability of engineered T cells to recognize intended tumor-associated targets is crucial for the therapeutic success, antigen-binding domains play an important role in shaping T-cell responses. Single-chain antibody and T-cell receptor fragments, natural ligands, repeat proteins, combinations of the above and universal tag-specific domains have all been used in the antigen-binding moiety of chimeric receptors. Here we outline the advantages and disadvantages of different domains, discuss the concepts of affinity and specificity, and highlight the recent progress of each targeting strategy.

Antigenic properties of the human immunodeficiency virus envelope glycoprotein gp120 on virions bound to target cells.

PubMed

Mengistu, Meron; Ray, Krishanu; Lewis, George K; DeVico, Anthony L

2015-03-01

The HIV-1 envelope glycoprotein, gp120, undergoes multiple molecular interactions and structural rearrangements during the course of host cell attachment and viral entry, which are being increasingly defined at the atomic level using isolated proteins. In comparison, antigenic markers of these dynamic changes are essentially unknown for single HIV-1 particles bound to target cells. Such markers should indicate how neutralizing and/or non-neutralizing antibodies might interdict infection by either blocking infection or sensitizing host cells for elimination by Fc-mediated effector function. Here we address this deficit by imaging fluorescently labeled CCR5-tropic HIV-1 pseudoviruses using confocal and superresolution microscopy to track the exposure of neutralizing and non-neutralizing epitopes as they appear on single HIV-1 particles bound to target cells. Epitope exposure was followed under conditions permissive or non-permissive for viral entry to delimit changes associated with virion binding from those associated with post-attachment events. We find that a previously unexpected array of gp120 epitopes is exposed rapidly upon target cell binding. This array comprises both neutralizing and non-neutralizing epitopes, the latter being hidden on free virions yet capable of serving as potent targets for Fc-mediated effector function. Under non-permissive conditions for viral entry, both neutralizing and non-neutralizing epitope exposures were relatively static over time for the majority of bound virions. Under entry-permissive conditions, epitope exposure patterns changed over time on subsets of virions that exhibited concurrent variations in virion contents. These studies reveal that bound virions are distinguished by a broad array of both neutralizing and non-neutralizing gp120 epitopes that potentially sensitize a freshly engaged target cell for destruction by Fc-mediated effector function and/or for direct neutralization at a post-binding step. The elucidation of

Differences in antigen presentation to MHC class I-and class II- restricted influenza virus-specific cytolytic T lymphocyte clones

PubMed Central

1986-01-01

We have examined requirements for antigen presentation to a panel of MHC class I-and class II-restricted, influenza virus-specific CTL clones by controlling the form of virus presented on the target cell surface. Both H-2K/D- and I region-restricted CTL recognize target cells exposed to infectious virus, but only the I region-restricted clones efficiently lysed histocompatible target cells pulsed with inactivated virus preparations. The isolated influenza hemagglutinin (HA) polypeptide also could sensitize target cells for recognition by class II-restricted, HA-specific CTL, but not by class I-restricted, HA- specific CTL. Inhibition of nascent viral protein synthesis abrogated the ability of target cells to present viral antigen relevant for class I-restricted CTL recognition. Significantly, presentation for class II- restricted recognition was unaffected in target cells exposed to preparations of either inactivated or infectious virus. This differential sensitivity suggested that these H-2I region-restricted CTL recognized viral polypeptides derived from the exogenously introduced virions, rather than viral polypeptides newly synthesized in the infected cell. In support of this contention, treatment of the target cells with the lysosomotropic agent chloroquine abolished recognition of infected target cells by class II-restricted CTL without diminishing class I-restricted recognition of infected target cells. Furthermore, when the influenza HA gene was introduced into target cells without exogenous HA polypeptide, the target cells that expressed the newly synthesized protein product of the HA gene were recognized only by H-2K/D-restricted CTL. These observations suggest that important differences may exist in requirements for antigen presentation between H-2K/D and H-2I region-restricted CTL. These differences may reflect the nature of the antigenic epitopes recognized by these two CTL subsets. PMID:3485173

[Research on the eventual cross-reactivity of anti-Wr(a) with various viral, bacterial and mycotic antigenes (author's transl)].

PubMed

Garelli, S; Valbonesi, M; Picerno, G; Vazzana, A

1978-09-01

Among the sera of 1011 blood donors, they have been collected 34 anti-Wr(a) antibodies. By IgG antiglobulin test, the titer was 1/8 or more in 21 sera. After absorption on viral, bacterial and mycotic antigens, the sera were still reactive with Wr(a) + red blood cells. These results show that no tested antigen is cross-reactive with Wr(a) antigen. However, the AA. suggest that the research of a widley diffused antigen, cross-reactive with Wr(a) + red blood cells, is a valuable approach to the problem of IgG anti-Wr(a) antibodies in normal, never transfused blood donors.

Targeted DNA Mutagenesis for the Cure of Chronic Viral Infections

PubMed Central

Schiffer, Joshua T.; Aubert, Martine; Weber, Nicholas D.; Mintzer, Esther; Stone, Daniel

2012-01-01

Human immunodeficiency virus type 1 (HIV-1), hepatitis B virus (HBV), and herpes simplex virus (HSV) have been incurable to date because effective antiviral therapies target only replicating viruses and do not eradicate latently integrated or nonreplicating episomal viral genomes. Endonucleases that can target and cleave critical regions within latent viral genomes are currently in development. These enzymes are being engineered with high specificity such that off-target binding of cellular DNA will be absent or minimal. Imprecise nonhomologous-end-joining (NHEJ) DNA repair following repeated cleavage at the same critical site may permanently disrupt translation of essential viral proteins. We discuss the benefits and drawbacks of three types of DNA cleavage enzymes (zinc finger endonucleases, transcription activator-like [TAL] effector nucleases [TALENs], and homing endonucleases [also called meganucleases]), the development of delivery vectors for these enzymes, and potential obstacles for successful treatment of chronic viral infections. We then review issues regarding persistence of HIV-1, HBV, and HSV that are relevant to eradication with genome-altering approaches. PMID:22718830

Rapid viral diagnosis of acute respiratory infections: comparison of enzyme-linked immunosorbent assay and the immunofluorescence technique for detection of viral antigens in nasopharyngeal secretions.

PubMed Central

Grandien, M; Pettersson, C A; Gardner, P S; Linde, A; Stanton, A

1985-01-01

Nasopharyngeal secretions from adults and children were obtained in Stockholm, Sweden, for routine diagnosis of influenza A virus, influenza B virus, respiratory syncytial (RS) virus, parainfluenza type 3 virus, and adenovirus infections by demonstration of viral antigens directly in the specimens. The cells in nasopharyngeal secretions were pelleted by centrifugation for preparation of cell deposits for diagnosis by the immunofluorescence technique (IF) in London, England, and in Stockholm, whereas the supernatants were used to diagnose infection by the enzyme-linked immunosorbent assay (ELISA) in Stockholm. Titrations of the various purified viruses showed that ELISA could detect viral antigens in amounts corresponding to 1 to 10 ng of virus protein per test well. In a series of 73 specimens tested for influenza A, RS, and parainfluenza type 3 viruses by IF in London and by ELISA in Stockholm, 15 of 18 RS, 14 of 15 influenza A, and 2 of 2 parainfluenza type 3 viral infections were diagnosed by ELISA as compared with IF, giving sensitivities for RS and influenza A viral diagnosis of 83 and 93%, respectively, and a specificity of 100%. In another series of specimens from 35 patients tested for influenza B virus and adenovirus, five influenza B virus and four adenovirus infections were diagnosed by both methods; one additional influenza B infection was detected only by IF and another only by ELISA. Comparisons of diagnostic results between the two methods performed in Stockholm gave nonagreement of results for 37 of 1,593 tests (2.5%) for the five viruses. The conclusion reached was that the described ELISA, although a satisfactory test, had somewhat less sensitivity than did IF for the detection of respiratory viral infections. This could possibly be explained by unnecessary dilutions of specimens at the time of collection; transportation, processing, and storage of specimens were less complicated than for IF. PMID:2997270

Surface-enhanced Raman scattering (SERS) detection of multiple viral antigens using magnetic capture of SERS-active nanoparticles

USDA-ARS?s Scientific Manuscript database

A highly sensitive immunoassay based on surface-enhanced Raman scattering (SERS) spectroscopy has been developed for multiplex detection of surface envelope and capsid antigens of the viral zoonotic pathogens West Nile virus (WNV) and Rift Valley fever virus (RVFV). Detection was mediated by antibo...

IT-25DEVELOPMENTALLY REGULATED ANTIGENS FOR IMMUNOLOGIC TARGETING OF MEDULLOBLASTOMA SUBTYPES

PubMed Central

Pham, Christina; Flores, Catherine; Pei, Yanxin; Wechsler-Reya, Robert; Mitchell, Duane

2014-01-01

INTRODUCTION: Medulloblastoma (MB) remains incurable in one third of patients despite aggressive multi-modality standard therapies. Immunotherapy presents a promising alternative by specifically targeting cancer cells. To date, there have been no successful immunologic applications targeting MB. Emerging evidence from integrated genomic studies has suggested MB variants arise from deregulation of pathways affecting proliferation of progenitor cell populations within the developing cerebellum. Using total embryonic RNA as a source of tumor rejection antigens is attractive because it can be delivered as a single vaccine, target both known and unknown fetal proteins, and can be refined to preferentially treat distinct MB subtypes. METHODS: We have created two transplantable, syngeneic animal MB models recapitulating human SHH and Group 3 variants to investigate the immunologic targeting of different MB subtypes. We generated T cells specific to the developing mouse cerebellum (P5) and tested their reactivity to target cells pulsed with total RNA from two MB subtypes and the normal brain. Immune responses were evaluated by measuring cytokine secretion following re-stimulation of activated T cells with both normal and tumor cell targets. In vivo antitumor efficacy was also tested in survival studies of intracranial tumor-bearing animals. RESULTS: We generated T cells specific to the developing cerebellum in vitro, confirming the immunogenicity of developmentally regulated antigens. Additionally, we have shown that developmental antigen-specific T cells produce high levels of Th1-type cytokines in response to tumor cells of two immunologically distinct subtypes of MB. Interestingly, developmental antigen specific T cells do not show cross reactivity with the normal brain or subsequent stages of the developing brain after P5. Targeting developmental antigens also conferred a significant survival benefit in a treatment model of Group 3 tumor bearing animals. CONCLUSIONS

A compound chimeric antigen receptor strategy for targeting multiple myeloma.

PubMed

Chen, K H; Wada, M; Pinz, K G; Liu, H; Shuai, X; Chen, X; Yan, L E; Petrov, J C; Salman, H; Senzel, L; Leung, E L H; Jiang, X; Ma, Y

2018-02-01

Current clinical outcomes using chimeric-antigen receptors (CARs) against multiple myeloma show promise in the eradication of bulk disease. However, these anti-BCMA (CD269) CARs observe relapse as a common phenomenon after treatment due to the reemergence of either antigen-positive or -negative cells. Hence, the development of improvements in CAR design to target antigen loss and increase effector cell persistency represents a critical need. Here, we report on the anti-tumor activity of a CAR T-cell possessing two complete and independent CAR receptors against the multiple myeloma antigens BCMA and CS1. We determined that the resulting compound CAR (cCAR) T-cell possesses consistent, potent and directed cytotoxicity against each target antigen population. Using multiple mouse models of myeloma and mixed cell populations, we are further able to show superior in vivo survival by directed cytotoxicity against multiple populations compared to a single-expressing CAR T-cell. These findings indicate that compound targeting of BCMA and CS1 on myeloma cells can potentially be an effective strategy for augmenting the response against myeloma bulk disease and for initiation of broader coverage CAR therapy.

Cas9 specifies functional viral targets during CRISPR-Cas adaptation.

PubMed

Heler, Robert; Samai, Poulami; Modell, Joshua W; Weiner, Catherine; Goldberg, Gregory W; Bikard, David; Marraffini, Luciano A

2015-03-12

Clustered regularly interspaced short palindromic repeat (CRISPR) loci and their associated (Cas) proteins provide adaptive immunity against viral infection in prokaryotes. Upon infection, short phage sequences known as spacers integrate between CRISPR repeats and are transcribed into small RNA molecules that guide the Cas9 nuclease to the viral targets (protospacers). Streptococcus pyogenes Cas9 cleavage of the viral genome requires the presence of a 5'-NGG-3' protospacer adjacent motif (PAM) sequence immediately downstream of the viral target. It is not known whether and how viral sequences flanked by the correct PAM are chosen as new spacers. Here we show that Cas9 selects functional spacers by recognizing their PAM during spacer acquisition. The replacement of cas9 with alleles that lack the PAM recognition motif or recognize an NGGNG PAM eliminated or changed PAM specificity during spacer acquisition, respectively. Cas9 associates with other proteins of the acquisition machinery (Cas1, Cas2 and Csn2), presumably to provide PAM-specificity to this process. These results establish a new function for Cas9 in the genesis of prokaryotic immunological memory.

Viral vector-based influenza vaccines.

PubMed

de Vries, Rory D; Rimmelzwaan, Guus F

2016-11-01

Antigenic drift of seasonal influenza viruses and the occasional introduction of influenza viruses of novel subtypes into the human population complicate the timely production of effective vaccines that antigenically match the virus strains that cause epidemic or pandemic outbreaks. The development of game-changing vaccines that induce broadly protective immunity against a wide variety of influenza viruses is an unmet need, in which recombinant viral vectors may provide. Use of viral vectors allows the delivery of any influenza virus antigen, or derivative thereof, to the immune system, resulting in the optimal induction of virus-specific B- and T-cell responses against this antigen of choice. This systematic review discusses results obtained with vectored influenza virus vaccines and advantages and disadvantages of the currently available viral vectors.

Effects of mutations within the SV40 large T antigen ATPase/p53 binding domain on viral replication and transformation.

PubMed

Peden, K W; Srinivasan, A; Vartikar, J V; Pipas, J M

1998-01-01

The simian virus 40 (SV40) large T antigen is a 708 amino-acid protein possessing multiple biochemical activities that play distinct roles in productive infection or virus-induced cell transformation. The carboxy-terminal portion of T antigen includes a domain that carries the nucleotide binding and ATPase activities of the protein, as well as sequences required for T antigen to associate with the cellular tumor suppressor p53. Consequently this domain functions both in viral DNA replication and cellular transformation. We have generated a collection of SV40 mutants with amino-acid deletions, insertions or substitutions in specific domains of the protein. Here we report the properties of nine mutants with single or multiple substitutions between amino acids 402 and 430, a region thought to be important for both the p53 binding and ATPase functions. The mutants were examined for the ability to produce infectious progeny virions, replicate viral DNA in vivo, perform in trans complementation tests, and transform established cell lines. Two of the mutants exhibited a wild-type phenotype in all these tests. The remaining seven mutants were defective for plaque formation and viral DNA replication, but in each case these defects could be complemented by a wild-type T antigen supplied in trans. One of these replication-defective mutants efficiently transformed the REF52 and C3H10T1/2 cell lines as assessed by the dense-focus assay. The remaining six mutants were defective for transforming REF52 cells and transformed the C3H10T1/2 line with a reduced efficiency. The ability of mutant T antigen to transform REF52 cells correlated with their ability to induce increased levels of p53.

Pros and Cons of Antigen-Presenting Cell Targeted Tumor Vaccines.

PubMed

Goyvaerts, Cleo; Breckpot, Karine

2015-01-01

In therapeutic antitumor vaccination, dendritic cells play the leading role since they decide if, how, when, and where a potent antitumor immune response will take place. Since the disentanglement of the complexity and merit of different antigen-presenting cell subtypes, antitumor immunotherapeutic research started to investigate the potential benefit of targeting these subtypes in situ. This review will discuss which antigen-presenting cell subtypes are at play and how they have been targeted and finally question the true meaning of targeting antitumor-based vaccines.

Viral vector-based influenza vaccines

PubMed Central

de Vries, Rory D.; Rimmelzwaan, Guus F.

2016-01-01

ABSTRACT Antigenic drift of seasonal influenza viruses and the occasional introduction of influenza viruses of novel subtypes into the human population complicate the timely production of effective vaccines that antigenically match the virus strains that cause epidemic or pandemic outbreaks. The development of game-changing vaccines that induce broadly protective immunity against a wide variety of influenza viruses is an unmet need, in which recombinant viral vectors may provide. Use of viral vectors allows the delivery of any influenza virus antigen, or derivative thereof, to the immune system, resulting in the optimal induction of virus-specific B- and T-cell responses against this antigen of choice. This systematic review discusses results obtained with vectored influenza virus vaccines and advantages and disadvantages of the currently available viral vectors. PMID:27455345

A Modeling and Experimental Investigation of the Effects of Antigen Density, Binding Affinity, and Antigen Expression Ratio on Bispecific Antibody Binding to Cell Surface Targets*

PubMed Central

Rhoden, John J.; Dyas, Gregory L.

2016-01-01

Despite the increasing number of multivalent antibodies, bispecific antibodies, fusion proteins, and targeted nanoparticles that have been generated and studied, the mechanism of multivalent binding to cell surface targets is not well understood. Here, we describe a conceptual and mathematical model of multivalent antibody binding to cell surface antigens. Our model predicts that properties beyond 1:1 antibody:antigen affinity to target antigens have a strong influence on multivalent binding. Predicted crucial properties include the structure and flexibility of the antibody construct, the target antigen(s) and binding epitope(s), and the density of antigens on the cell surface. For bispecific antibodies, the ratio of the expression levels of the two target antigens is predicted to be critical to target binding, particularly for the lower expressed of the antigens. Using bispecific antibodies of different valencies to cell surface antigens including MET and EGF receptor, we have experimentally validated our modeling approach and its predictions and observed several nonintuitive effects of avidity related to antigen density, target ratio, and antibody affinity. In some biological circumstances, the effect we have predicted and measured varied from the monovalent binding interaction by several orders of magnitude. Moreover, our mathematical framework affords us a mechanistic interpretation of our observations and suggests strategies to achieve the desired antibody-antigen binding goals. These mechanistic insights have implications in antibody engineering and structure/activity relationship determination in a variety of biological contexts. PMID:27022022

A Modeling and Experimental Investigation of the Effects of Antigen Density, Binding Affinity, and Antigen Expression Ratio on Bispecific Antibody Binding to Cell Surface Targets.

PubMed

Rhoden, John J; Dyas, Gregory L; Wroblewski, Victor J

2016-05-20

Despite the increasing number of multivalent antibodies, bispecific antibodies, fusion proteins, and targeted nanoparticles that have been generated and studied, the mechanism of multivalent binding to cell surface targets is not well understood. Here, we describe a conceptual and mathematical model of multivalent antibody binding to cell surface antigens. Our model predicts that properties beyond 1:1 antibody:antigen affinity to target antigens have a strong influence on multivalent binding. Predicted crucial properties include the structure and flexibility of the antibody construct, the target antigen(s) and binding epitope(s), and the density of antigens on the cell surface. For bispecific antibodies, the ratio of the expression levels of the two target antigens is predicted to be critical to target binding, particularly for the lower expressed of the antigens. Using bispecific antibodies of different valencies to cell surface antigens including MET and EGF receptor, we have experimentally validated our modeling approach and its predictions and observed several nonintuitive effects of avidity related to antigen density, target ratio, and antibody affinity. In some biological circumstances, the effect we have predicted and measured varied from the monovalent binding interaction by several orders of magnitude. Moreover, our mathematical framework affords us a mechanistic interpretation of our observations and suggests strategies to achieve the desired antibody-antigen binding goals. These mechanistic insights have implications in antibody engineering and structure/activity relationship determination in a variety of biological contexts. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

An Exosome-Based Vaccine Platform Imparts Cytotoxic T Lymphocyte Immunity Against Viral Antigens.

PubMed

Anticoli, Simona; Manfredi, Francesco; Chiozzini, Chiara; Arenaccio, Claudia; Olivetta, Eleonora; Ferrantelli, Flavia; Capocefalo, Antonio; Falcone, Emiliana; Ruggieri, Anna; Federico, Maurizio

2018-04-01

Exosomes are 50-150 nm sized nanovesicles released by all eukaryotic cells. The authors very recently described a method to engineer exosomes in vivo with the E7 protein of Human Papilloma Virus (HPV). This technique consists in the intramuscular injection of a DNA vector expressing HPV-E7 fused at the C-terminus of an exosome-anchoring protein, that is, Nef mut , the authors previously characterized for its high levels of incorporation in exosomes. In this configuration, the ≈11 kDa E7 protein elicited a both strong and effective antigen-specific cytotoxic T lymphocyte (CTL) immunity. Attempting to establish whether this method could have general applicability, the authors expanded the immunogenicity studies toward an array of viral products of various origin and size including Ebola Virus VP24, VP40 and NP, Influenza Virus NP, Crimean-Congo Hemorrhagic Fever NP, West Nile Virus NS3, and Hepatitis C Virus NS3. All antigens appeared stable upon fusion with Nef mut , and are uploaded in exosomes at levels comparable to Nef mut . When injected in mice, DNA vectors expressing the diverse fusion products elicited a well detectable antigen-specific CD8 + T cell response associating with a cytotoxic activity potent enough to kill peptide-loaded and/or antigen-expressing syngeneic cells. These data definitely proven both effectiveness and flexibility of this innovative CTL vaccine platform. © 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Targeting of a Nuclease to Murine Leukemia Virus Capsids Inhibits Viral Multiplication

NASA Astrophysics Data System (ADS)

Natsoulis, Georges; Seshaiah, Partha; Federspiel, Mark J.; Rein, Alan; Hughes, Stephen H.; Boeke, Jef D.

1995-01-01

Capsid-targeted viral inactivation is an antiviral strategy in which toxic fusion proteins are targeted to virions, where they inhibit viral multiplication by destroying viral components. These fusion proteins consist of a virion structural protein moiety and an enzymatic moiety such as a nuclease. Such fusion proteins can severely inhibit transposition of yeast retrotransposon Ty1, an element whose transposition mechanistically resembles retroviral multiplication. We demonstrate that expression of a murine retrovirus capsid-staphylococcal nuclease fusion protein inhibits multiplication of the corresponding murine leukemia virus by 30- to 100-fold. Staphylococcal nuclease is apparently inactive intracellularly and hence nontoxic to the host cell, but it is active extracellularly because of its requirement for high concentrations of Ca2+ ions. Virions assembled in and shed from cells expressing the fusion protein contain very small amounts of intact viral RNA, as would be predicted for nuclease-mediated inhibition of viral multiplication.

Nuclear targeting of viral and non-viral DNA.

PubMed

Chowdhury, E H

2009-07-01

The nuclear envelope presents a major barrier to transgene delivery and expression using a non-viral vector. Virus is capable of overcoming the barrier to deliver their genetic materials efficiently into the nucleus by virtue of the specialized protein components with the unique amino acid sequences recognizing cellular nuclear transport machinery. However, considering the safety issues in the clinical gene therapy for treating critical human diseases, non-viral systems are highly promising compared with their viral counterparts. This review summarizes the progress on exploring the nuclear traffic mechanisms for the prominent viral vectors and the technological innovations for the nuclear delivery of non-viral DNA by mimicking those natural processes evolved for the viruses as well as for many cellular proteins.

T LYMPHOCYTES TARGETING NATIVE RECEPTORS

PubMed Central

Rooney, Cliona M; Leen, Ann M; Vera, Juan F; Heslop, Helen E

2013-01-01

Summary The adoptive transfer of T cells specific for native tumor antigens (TAs) is an increasingly popular cancer treatment option because of the ability of these cells to discriminate between normal and tumor tissues and corresponding lack of short or long-term toxicities. Infusions of antigen-specific CD4+ and CD8+ T cells targeting viral antigens derived from Epstein Barr virus (EBV) induce sustained complete tumor remissions in patients with highly immunogenic tumor’s such as post-transplant lymphoproliferative disease, although resistance occurred when the infused T-cell population had restricted antigen specificity. T cells specific for EBV antigens have also produced complete remissions of EBV-positive nasopharyngeal carcinomas and lymphomas developing in immunocompetent individuals, even though in these patients tumor survival is dependent on their ability to evade T-cell immunity. Adapting this strategy to non-viral tumors is more challenging, as the target antigens expressed are less immunogenic and the tumors lack the potent danger signals that are characteristic of viruses. The goals of current studies are to define conditions that promote expansion of antigen-specific T cells ex vivo and to ensure their in vivo persistence and survival by combining with maneuvers such as lymphodepletion, checkpoint inhibition, cytokine infusions, or genetic manipulations. More pragmatic goals are to streamline manufacturing to facilitate the transition of these therapies to late phase trials and to evaluate closely histocompatibility antigen (HLA)-matched banked antigen-specific T-cells so that T-cell therapies can be made more broadly available. PMID:24329788

VIRmiRNA: a comprehensive resource for experimentally validated viral miRNAs and their targets.

PubMed

Qureshi, Abid; Thakur, Nishant; Monga, Isha; Thakur, Anamika; Kumar, Manoj

2014-01-01

Viral microRNAs (miRNAs) regulate gene expression of viral and/or host genes to benefit the virus. Hence, miRNAs play a key role in host-virus interactions and pathogenesis of viral diseases. Lately, miRNAs have also shown potential as important targets for the development of novel antiviral therapeutics. Although several miRNA and their target repositories are available for human and other organisms in literature, but a dedicated resource on viral miRNAs and their targets are lacking. Therefore, we have developed a comprehensive viral miRNA resource harboring information of 9133 entries in three subdatabases. This includes 1308 experimentally validated miRNA sequences with their isomiRs encoded by 44 viruses in viral miRNA ' VIRMIRNA: ' and 7283 of their target genes in ' VIRMIRTAR': . Additionally, there is information of 542 antiviral miRNAs encoded by the host against 24 viruses in antiviral miRNA ' AVIRMIR': . The web interface was developed using Linux-Apache-MySQL-PHP (LAMP) software bundle. User-friendly browse, search, advanced search and useful analysis tools are also provided on the web interface. VIRmiRNA is the first specialized resource of experimentally proven virus-encoded miRNAs and their associated targets. This database would enhance the understanding of viral/host gene regulation and may also prove beneficial in the development of antiviral therapeutics. Database URL: http://crdd.osdd.net/servers/virmirna. © The Author(s) 2014. Published by Oxford University Press.

Antibody-Antigen-Adjuvant Conjugates Enable Co-Delivery of Antigen and Adjuvant to Dendritic Cells in Cis but Only Have Partial Targeting Specificity

PubMed Central

Abuknesha, Ram; Uematsu, Satoshi; Akira, Shizuo; Nestle, Frank O.; Diebold, Sandra S.

2012-01-01

Antibody-antigen conjugates, which promote antigen-presentation by dendritic cells (DC) by means of targeted delivery of antigen to particular DC subsets, represent a powerful vaccination approach. To ensure immunity rather than tolerance induction the co-administration of a suitable adjuvant is paramount. However, co-administration of unlinked adjuvant cannot ensure that all cells targeted by the antibody conjugates are appropriately activated. Furthermore, antigen-presenting cells (APC) that do not present the desired antigen are equally strongly activated and could prime undesired responses against self-antigens. We, therefore, were interested in exploring targeted co-delivery of antigen and adjuvant in cis in form of antibody-antigen-adjuvant conjugates for the induction of anti-tumour immunity. In this study, we report on the assembly and characterization of conjugates consisting of DEC205-specific antibody, the model antigen ovalbumin (OVA) and CpG oligodeoxynucleotides (ODN). We show that such conjugates are more potent at inducing cytotoxic T lymphocyte (CTL) responses than control conjugates mixed with soluble CpG. However, our study also reveals that the nucleic acid moiety of such antibody-antigen-adjuvant conjugates alters their binding and uptake and allows delivery of the antigen and the adjuvant to cells partially independently of DEC205. Nevertheless, antibody-antigen-adjuvant conjugates are superior to antibody-free antigen-adjuvant conjugates in priming CTL responses and efficiently induce anti-tumour immunity in the murine B16 pseudo-metastasis model. A better understanding of the role of the antibody moiety is required to inform future conjugate vaccination strategies for efficient induction of anti-tumour responses. PMID:22808118

Checkpoint Blockade Cancer Immunotherapy Targets Tumour-Specific Mutant Antigens

PubMed Central

Gubin, Matthew M.; Zhang, Xiuli; Schuster, Heiko; Caron, Etienne; Ward, Jeffrey P.; Noguchi, Takuro; Ivanova, Yulia; Hundal, Jasreet; Arthur, Cora D.; Krebber, Willem-Jan; Mulder, Gwenn E.; Toebes, Mireille; Vesely, Matthew D.; Lam, Samuel S.K.; Korman, Alan J.; Allison, James P.; Freeman, Gordon J.; Sharpe, Arlene H.; Pearce, Erika L.; Schumacher, Ton N.; Aebersold, Ruedi; Rammensee, Hans-Georg; Melief, Cornelis J. M.; Mardis, Elaine R.; Gillanders, William E.; Artyomov, Maxim N.; Schreiber, Robert D.

2014-01-01

The immune system plays key roles in determining the fate of developing cancers by not only functioning as a tumour promoter facilitating cellular transformation, promoting tumour growth and sculpting tumour cell immunogenicity1–6, but also as an extrinsic tumour suppressor that either destroys developing tumours or restrains their expansion1,2,7. Yet clinically apparent cancers still arise in immunocompetent individuals in part as a consequence of cancer induced immunosuppression. In many individuals, immunosuppression is mediated by Cytotoxic T-Lymphocyte Associated Antigen-4 (CTLA-4) and Programmed Death-1 (PD-1), two immunomodulatory receptors expressed on T cells8,9. Monoclonal antibody (mAb) based therapies targeting CTLA-4 and/or PD-1 (checkpoint blockade) have yielded significant clinical benefits—including durable responses—to patients with different malignancies10–13. However, little is known about the identity of the tumour antigens that function as the targets of T cells activated by checkpoint blockade immunotherapy and whether these antigens can be used to generate vaccines that are highly tumour-specific. Herein, we use genomics and bioinformatics approaches to identify tumour-specific mutant proteins as a major class of T cell rejection antigens following αPD-1 and/or αCTLA-4 therapy of mice bearing progressively growing sarcomas and show that therapeutic synthetic long peptide (SLP) vaccines incorporating these mutant epitopes induce tumour rejection comparably to checkpoint blockade immunotherapy. Whereas, mutant tumour antigen-specific T cells are present in progressively growing tumours, they are reactivated following treatment with αPD-1- and/or αCTLA-4 and display some overlapping but mostly treatment-specific transcriptional profiles rendering them capable of mediating tumour rejection. These results reveal that tumour-specific mutant antigens (TSMA) are not only important targets of checkpoint blockade therapy but also can be

Prostate Specific Membrane Antigen (PSMA) Targeted Bio-orthogonal Therapy for Metastatic Prostate Cancer

DTIC Science & Technology

2017-10-01

AWARD NUMBER: W81XWH-16-1-0595 TITLE: Prostate-Specific Membrane Antigen (PSMA) Targeted Bio -orthogonal Therapy for Metastatic Prostate Cancer...Sep 2016 - 14 Sep 2017 4. TITLE AND SUBTITLE Prostate-Specific Membrane Antigen (PSMA) Targeted Bio -orthogonal Therapy for Metastatic Prostate

Cell-permeable nanobodies for targeted immunolabelling and antigen manipulation in living cells

NASA Astrophysics Data System (ADS)

Herce, Henry D.; Schumacher, Dominik; Schneider, Anselm F. L.; Ludwig, Anne K.; Mann, Florian A.; Fillies, Marion; Kasper, Marc-André; Reinke, Stefan; Krause, Eberhard; Leonhardt, Heinrich; Cardoso, M. Cristina; Hackenberger, Christian P. R.

2017-08-01

Functional antibody delivery in living cells would enable the labelling and manipulation of intracellular antigens, which constitutes a long-thought goal in cell biology and medicine. Here we present a modular strategy to create functional cell-permeable nanobodies capable of targeted labelling and manipulation of intracellular antigens in living cells. The cell-permeable nanobodies are formed by the site-specific attachment of intracellularly stable (or cleavable) cyclic arginine-rich cell-penetrating peptides to camelid-derived single-chain VHH antibody fragments. We used this strategy for the non-endocytic delivery of two recombinant nanobodies into living cells, which enabled the relocalization of the polymerase clamp PCNA (proliferating cell nuclear antigen) and tumour suppressor p53 to the nucleolus, and thereby allowed the detection of protein-protein interactions that involve these two proteins in living cells. Furthermore, cell-permeable nanobodies permitted the co-transport of therapeutically relevant proteins, such as Mecp2, into the cells. This technology constitutes a major step in the labelling, delivery and targeted manipulation of intracellular antigens. Ultimately, this approach opens the door towards immunostaining in living cells and the expansion of immunotherapies to intracellular antigen targets.

A viral, transporter associated with antigen processing (TAP)-independent, high affinity ligand with alternative interactions endogenously presented by the nonclassical human leukocyte antigen E class I molecule.

PubMed

Lorente, Elena; Infantes, Susana; Abia, David; Barnea, Eilon; Beer, Ilan; García, Ruth; Lasala, Fátima; Jiménez, Mercedes; Mir, Carmen; Morreale, Antonio; Admon, Arie; López, Daniel

2012-10-12

The transporter associated with antigen processing (TAP) enables the flow of viral peptides generated in the cytosol by the proteasome and other proteases to the endoplasmic reticulum, where they complex with nascent human leukocyte antigen (HLA) class I. Later, these peptide-HLA class I complexes can be recognized by CD8(+) lymphocytes. Cancerous cells and infected cells in which TAP is blocked, as well as individuals with unusable TAP complexes, are able to present peptides on HLA class I by generating them through TAP-independent processing pathways. Here, we identify a physiologically processed HLA-E ligand derived from the D8L protein in TAP-deficient vaccinia virus-infected cells. This natural high affinity HLA-E class I ligand uses alternative interactions to the anchor motifs previously described to be presented on nonclassical HLA class I molecules. This octameric peptide was also presented on HLA-Cw1 with similar binding affinity on both classical and nonclassical class I molecules. In addition, this viral peptide inhibits HLA-E-mediated cytolysis by natural killer cells. Comparison between the amino acid sequences of the presenting HLA-E and HLA-Cw1 alleles revealed a shared structural motif in both HLA class molecules, which could be related to their observed similar cross-reactivity affinities. This motif consists of several residues located on the floor of the peptide-binding site. These data expand the role of HLA-E as an antigen-presenting molecule.

Is HCV core antigen a reliable marker of viral load? An evaluation of HCV core antigen automated immunoassay

PubMed Central

Hadziyannis, Emilia; Minopetrou, Martha; Georgiou, Anastasia; Spanou, Fotini; Koskinas, John

2013-01-01

Background Hepatitis C viral (HCV) load detection and quantification is routinely accomplished by HCV RNA measurement, an expensive but essential test, both for the diagnosis and treatment of chronic hepatitis C (CHC). HCV core antigen (Ag) testing has been suggested as an attractive alternative to molecular diagnostics. The aim of the study was to evaluate an automated chemiluminescent immunoassay (CLIA) for HCV core Ag measurement in comparison to quantitative HCV RNA determination. Methods HCV Ag was measured in 105 anti-HCV positive patients, from which 89 were HCV RNA positive with CHC and 16 HCV RNA negative after spontaneous HCV clearance. Viral load was quantified with branched DNA (bDNA, Versant, Siemens). Sera were stored at -70°C and then tested with the Architect HCV Ag test (Abbott Laboratories), a two-step CLIA assay, with high throughput and minimal handling of the specimens. Statistical analysis was performed on logarithmically transformed values. Results HCV-Ag was detectable and quantifiable in 83/89 and in grey zone in 4/89 HCV RNA positive sera. HCV-Ag was undetectable in all 16 HCV RNA negative samples. The sample with the lowest viral load that tested positive for HCV-Ag contained 1200 IU/mL HCV RNA. There was a positive correlation between HCV RNA and HCV-Ag (r=0.89). The HCV RNA/ HCV Ag ratio varied from 1.5 to 3.25. Conclusion The HCV core Ag is an easy test with comparable sensitivity (>90%) and satisfactory correlation with the HCV RNA bDNA assay. Its role in diagnostics and other clinical applications has to be determined based on cost effectiveness. PMID:24714621

A multi-scale mathematical modeling framework to investigate anti-viral therapeutic opportunities in targeting HIV-1 accessory proteins

PubMed Central

Suryawanshi, Gajendra W.; Hoffmann, Alexander

2015-01-01

Human immunodeficiency virus-1 (HIV-1) employs accessory proteins to evade innate immune responses by neutralizing the anti-viral activity of host restriction factors. Apolipoprotein B mRNA-editing enzyme 3G (APOBEC3G, A3G) and bone marrow stromal cell antigen 2 (BST2) are host resistance factors that potentially inhibit HIV-1 infection. BST2 reduces viral production by tethering budding HIV-1 particles to virus producing cells, while A3G inhibits the reverse transcription (RT) process and induces viral genome hypermutation through cytidine deamination, generating fewer replication competent progeny virus. Two HIV-1 proteins counter these cellular restriction factors: Vpu, which reduces surface BST2, and Vif, which degrades cellular A3G. The contest between these host and viral proteins influences whether HIV-1 infection is established and progresses towards AIDS. In this work, we present an age-structured multi-scale viral dynamics model of in vivo HIV-1 infection. We integrated the intracellular dynamics of anti-viral activity of the host factors and their neutralization by HIV-1 accessory proteins into the virus/cell population dynamics model. We calculate the basic reproductive ratio (Ro) as a function of host-viral protein interaction coefficients, and numerically simulated the multi-scale model to understand HIV-1 dynamics following host factor-induced perturbations. We found that reducing the influence of Vpu triggers a drop in Ro, revealing the impact of BST2 on viral infection control. Reducing Vif’s effect reveals the restrictive efficacy of A3G in blocking RT and in inducing lethal hypermutations, however, neither of these factors alone is sufficient to fully restrict HIV-1 infection. Interestingly, our model further predicts that BST2 and A3G function synergistically, and delineates their relative contribution in limiting HIV-1 infection and disease progression. We provide a robust modeling framework for devising novel combination therapies that

Immunization with viral antigens: Infectious haematopoietic necrosis

USGS Publications Warehouse

Winton, J.R.; Midtlyng, Paul J.; Brown, F.

1997-01-01

Infectious haematopoietic necrosis (IHN) is one of the most important viral diseases of salmonids, especially among juvenile fish where losses can be high. For over 20 years, researchers have tested a variety of preparations for control of IHN. Early vaccines consisted of killed virus and were effective when delivered by injection, but too costly to be practical on a large scale. Attenuated vaccines were developed by serial passage in cell culture and by monoclonal antibody selection. These offered excellent protection and were cost-effective, but residual virulence and uncertainty about their effects on other aquatic species made them poor candidates for licensing. Subunit vaccines using part of the IHNV glycoprotein gene cloned into E. coli or into an attenuated strain of A. salmonicida have been tested, appeared safe and were inexpensive. These vaccines were reported to provide some protection when delivered by immersion. Information on the location of antigenic sites on the glycoprotein led to trials using synthetic peptides, but these did not seem to be economically viable. Recently, plasmid vectors encoding the glycoprotein gene under control of a cytomegalovirus promoter were developed for genetic immunization. The constructs were highly protective when delivered by injection, but a more practical delivery system is needed. Thus, while several vaccine strategies have been tried in order to stimulate specific immunity against IHN, more research is needed to develop a commercially viable product for control of this important disease.

Shared target antigens on cancer cells and tissue stem cells: go or no-go for CAR T cells?

PubMed

Hombach, Andreas A; Abken, Hinrich

2017-02-01

Adoptive therapy with chimeric antigen receptor (CAR) T cells redirected towards CD19 produces remissions of B cell malignancies, however, it also eradicates healthy B cells sharing the target antigen. Such 'on-target off-tumor' toxicity raises serious safety concerns when the target antigen is also expressed by tissue stem cells, with the risk of lasting tissue destruction. Areas covered: We discuss CAR T cell targeting of activation antigens versus lineage associated antigens on the basis of recent experimental and animal data and the literature in the field. Expert commentary: Targeting an activation associated antigen which is transiently expressed by stem cells seems to be safe, like CAR T cells targeting CD30 spare CD30 + hematopoietic stem and progenitor cells while eliminating CD30 + lymphoma cells, whereas targeting lineage associated antigens which increase in expression during cell maturation, like folate receptor-β and CD123, is of risk to destruct tissue stem cells.

Viral Capsid DNA Aptamer Conjugates as Multivalent Cell Targeting Vehicles

PubMed Central

Tong, Gary J.; Hsiao, Sonny C.; Carrico, Zachary M.; Francis, Matthew B.

2009-01-01

Nucleic acid aptamers offer significant potential as convenient and evolvable targeting groups for drug delivery. To attach them to the surface of a genome-free viral capsid carrier, an efficient oxidative coupling strategy has been developed. The method involves the periodate-mediated reaction of phenylene diamine substituted oligonucleotides with aniline groups installed on the outer surface of the capsid shells. Up to 60 DNA strands can be attached to each viral capsid with no apparent loss of base-pairing capabilities or protein stability. The ability of the capsids to bind specific cellular targets was demonstrated through the attachment of a 41-nucleotide sequence that targets a tyrosine kinase receptor on Jurkat T cells. After the installation of a fluorescent dye on the capsid interior, capsids bearing the cell-targeting sequence showed significant levels of binding to the cells relative to control samples. Colocalization experiments using confocal microscopy indicated that the capsids were endocytosed and trafficked to lysosomes for degradation. These observations suggest that aptamer-labeled capsids could be used for the targeted drug delivery of acid-labile prodrugs that would be preferentially released upon lysosomal acidification. PMID:19603808

A DNA Vaccine That Targets Hemagglutinin to Antigen-Presenting Cells Protects Mice against H7 Influenza

PubMed Central

Andersen, Tor Kristian; Zhou, Fan; Cox, Rebecca; Bogen, Bjarne

2017-01-01

ABSTRACT Zoonotic influenza H7 viral infections have a case fatality rate of about 40%. Currently, no or limited human to human spread has occurred, but we may be facing a severe pandemic threat if the virus acquires the ability to transmit between humans. Novel vaccines that can be rapidly produced for global distribution are urgently needed, and DNA vaccines may be the only type of vaccine that allows for the speed necessary to quench an emerging pandemic. Here, we constructed DNA vaccines encoding the hemagglutinin (HA) from influenza A/chicken/Italy/13474/99 (H7N1). In order to increase the efficacy of DNA vaccination, HA was targeted to either major histocompatibility complex class II molecules or chemokine receptors 1, 3, and 5 (CCR1/3/5) that are expressed on antigen-presenting cells (APC). A single DNA vaccination with APC-targeted HA significantly increased antibody levels in sera compared to nontargeted control vaccines. The antibodies were confirmed neutralizing in an H7 pseudotype-based neutralization assay. Furthermore, the APC-targeted vaccines increased the levels of antigen-specific cytotoxic T cells, and a single DNA vaccination could confer protection against a lethal challenge with influenza A/turkey/Italy/3889/1999 (H7N1) in mice. In conclusion, we have developed a vaccine that rapidly could contribute protection against a pandemic threat from avian influenza. IMPORTANCE Highly pathogenic avian influenza H7 constitute a pandemic threat that can cause severe illness and death in infected individuals. Vaccination is the main method of prophylaxis against influenza, but current vaccine strategies fall short in a pandemic situation due to a prolonged production time and insufficient production capabilities. In contrast, a DNA vaccine can be rapidly produced and deployed to prevent the potential escalation of a highly pathogenic influenza pandemic. We here demonstrate that a single DNA delivery of hemagglutinin from an H7 influenza could mediate full

Latency reversal and viral clearance to cure HIV-1

PubMed Central

Margolis, David M.; Garcia, J. Victor; Hazuda, Daria J.; Haynes, Barton F.

2016-01-01

Research toward a cure for human immunodeficiency virus type 1 (HIV-1) infection has joined prevention and treatment efforts in the global public health agenda. A major approach to HIV eradication envisions antiretroviral suppression, paired with targeted therapies to enforce the expression of viral antigen from quiescent HIV-1 genomes, and immunotherapies to clear latent infection. These strategies are targeted to lead to viral eradication—a cure for AIDS. Paired testing of latency reversal and clearance strategies has begun, but additional obstacles to HIV eradication may emerge. Nevertheless, there is reason for optimism that advances in long-acting antiretroviral therapy and HIV prevention strategies will contribute to efforts in HIV cure research and that the implementation of these efforts will synergize to markedly blunt the effect of the HIV pandemic on society. PMID:27463679

Prostate Cancer Relevant Antigens and Enzymes for Targeted Drug Delivery

PubMed Central

Barve, Ashutosh; Jin, Wei; Cheng, Kun

2014-01-01

Chemotherapy is one of the most widely used approaches in combating advanced prostate cancer, but its therapeutic efficacy is usually insufficient due to lack of specificity and associated toxicity. Lack of targeted delivery to prostate cancer cells is also the primary obstacles in achieving feasible therapeutic effect of other promising agents including peptide, protein, and nucleic acid. Consequently, there remains a critical need for strategies to increase the selectivity of anti-prostate cancer agents. This review will focus on various prostate cancer-specific antigens and enzymes that could be exploited for prostate cancer targeted drug delivery. Among various targeting strategies, active targeting is the most advanced approach to specifically deliver drugs to their designated cancer cells. In this approach, drug carriers are modified with targeting ligands that can specifically bind to prostate cancer-specific antigens. Moreover, there are several specific enzymes in the tumor microenvironment of prostate cancer that can be exploited for stimulus-responsive drug delivery systems. These systems can specifically release the active drug in the tumor microenvironment of prostate cancer, leading to enhanced tumor penetration efficiency. PMID:24878184

Universal Artificial Antigen Presenting Cells to Selectively Propagate T Cells Expressing Chimeric Antigen Receptor Independent of Specificity

PubMed Central

Rushworth, David; Jena, Bipulendu; Olivares, Simon; Maiti, Sourindra; Briggs, Neima; Somanchi, Srinivas; Dai, Jianliang; Lee, Dean; Cooper, Laurence J. N.

2014-01-01

T cells genetically modified to stably express immunoreceptors are being assessed for therapeutic potential in clinical trials. T cells expressing a chimeric antigen receptor (CAR) are endowed with a new specificity to target tumor-associated antigen (TAA) independent of major histocompatibility complex. Our approach to non-viral gene transfer in T cells uses ex vivo numeric expansion of CAR+ T cells on irradiated artificial antigen presenting cells (aAPC) bearing the targeted TAA. The requirement for aAPC to express a desired TAA limits the human application of CARs with multiple specificities when selective expansion through co-culture with feeder cells is sought. As an alternative to expressing individual TAAs on aAPC, we expressed one ligand that could activate CAR+ T cells for sustained proliferation independent of specificity. We expressed a CAR ligand (designated CARL) that binds the conserved IgG4 extracellular domain of CAR and demonstrated CARL+ aAPC propagate CAR+ T cells of multiple specificities. CARL avoids technical issues and costs associated with deploying clinical-grade aAPC for each TAA targeted by a given CAR. Employing CARL enables one aAPC to numerically expand all CAR+ T cells containing the IgG4 domain, and simplifies expansion, testing, and clinical translation of CAR+ T cells of any specificity. PMID:24714354

Fusion Protein Vaccines Targeting Two Tumor Antigens Generate Synergistic Anti-Tumor Effects

PubMed Central

Cheng, Wen-Fang; Chang, Ming-Cheng; Sun, Wei-Zen; Jen, Yu-Wei; Liao, Chao-Wei; Chen, Yun-Yuan; Chen, Chi-An

2013-01-01

Introduction Human papillomavirus (HPV) has been consistently implicated in causing several kinds of malignancies, and two HPV oncogenes, E6 and E7, represent two potential target antigens for cancer vaccines. We developed two fusion protein vaccines, PE(ΔIII)/E6 and PE(ΔIII)/E7 by targeting these two tumor antigens to test whether a combination of two fusion proteins can generate more potent anti-tumor effects than a single fusion protein. Materials and Methods In vivo antitumor effects including preventive, therapeutic, and antibody depletion experiments were performed. In vitro assays including intracellular cytokine staining and ELISA for Ab responses were also performed. Results PE(ΔIII)/E6+PE(ΔIII)/E7 generated both stronger E6 and E7-specific immunity. Only 60% of the tumor protective effect was observed in the PE(ΔIII)/E6 group compared to 100% in the PE(ΔIII)/E7 and PE(ΔIII)/E6+PE(ΔIII)/E7 groups. Mice vaccinated with the PE(ΔIII)/E6+PE(ΔIII)/E7 fusion proteins had a smaller subcutaneous tumor size than those vaccinated with PE(ΔIII)/E6 or PE(ΔIII)/E7 fusion proteins alone. Conclusion Fusion protein vaccines targeting both E6 and E7 tumor antigens generated more potent immunotherapeutic effects than E6 or E7 tumor antigens alone. This novel strategy of targeting two tumor antigens together can promote the development of cancer vaccines and immunotherapy in HPV-related malignancies. PMID:24058440

Multivalent glycopeptide dendrimers for the targeted delivery of antigens to dendritic cells.

PubMed

García-Vallejo, Juan J; Ambrosini, Martino; Overbeek, Annemieke; van Riel, Wilhelmina E; Bloem, Karien; Unger, Wendy W J; Chiodo, Fabrizio; Bolscher, Jan G; Nazmi, Kamran; Kalay, Hakan; van Kooyk, Yvette

2013-04-01

Dendritic cells are the most powerful type of antigen presenting cells. Current immunotherapies targeting dendritic cells have shown a relative degree of success but still require further improvement. One of the most important issues to solve is the efficiency of antigen delivery to dendritic cells in order to achieve an appropriate uptake, processing, and presentation to Ag-specific T cells. C-type lectins have shown to be ideal receptors for the targeting of antigens to dendritic cells and allow the use of their natural ligands - glycans - instead of antibodies. Amongst them, dendritic cell-specific ICAM-3-grabbing non-integrin (DC-SIGN) is an interesting candidate due to its biological properties and the availability of its natural carbohydrate ligands. Using Le(b)-conjugated poly(amido amine) (PAMAM) dendrimers we aimed to characterize the optimal level of multivalency necessary to achieve the desired internalization, lysosomal delivery, Ag-specific T cell proliferation, and cytokine response. Increasing DC-SIGN ligand multivalency directly translated in an enhanced binding, which might also be interesting for blocking purposes. Internalization, routing to lysosomal compartments, antigen presentation and cytokine response could be optimally achieved with glycopeptide dendrimers carrying 16-32 glycan units. This report provides the basis for the design of efficient targeting of peptide antigens for the immunotherapy of cancer, autoimmunity and infectious diseases. Copyright © 2012 Elsevier Ltd. All rights reserved.

Antibodies with 'Original Antigenic Sin' Properties Are Valuable Components of Secondary Immune Responses to Influenza Viruses.

PubMed

Linderman, Susanne L; Hensley, Scott E

2016-08-01

Human antibodies (Abs) elicited by influenza viruses often bind with a high affinity to past influenza virus strains, but paradoxically, do not bind to the viral strain actually eliciting the response. This phenomena is called 'original antigenic sin' (OAS) since this can occur at the expense of generating new de novo Abs. Here, we characterized the specificity and functionality of Abs elicited in mice that were sequentially exposed to two antigenically distinct H1N1 influenza virus strains. Many Abs elicited under these conditions had an OAS phenotype, in that they bound strongly to the viral strain used for the first exposure and very weakly to the viral strain used for the second exposure. We found that OAS and non-OAS Abs target the same general region of the influenza hemagglutinin protein and that B cells expressing these two types of Abs can be clonally-related. Surprisingly, although OAS Abs bound with very low affinities, some were able to effectively protect against an antigenically drifted viral strain following passive transfer in vivo. Taken together, our data indicate that OAS Abs share some level of cross-reactivity between priming and recall viral strains and that B cells producing these Abs can be protective when recalled into secondary immune responses.

Dual targeting of glioblastoma with chimeric antigen receptor-engineered natural killer cells overcomes heterogeneity of target antigen expression and enhances antitumor activity and survival.

PubMed

Genßler, Sabrina; Burger, Michael C; Zhang, Congcong; Oelsner, Sarah; Mildenberger, Iris; Wagner, Marlies; Steinbach, Joachim P; Wels, Winfried S

2016-04-01

Epidermal growth factor receptor (EGFR) and its mutant form EGFRvIII are overexpressed in a large proportion of glioblastomas (GBM). Immunotherapy with an EGFRvIII-specific vaccine has shown efficacy against GBM in clinical studies. However, immune escape by antigen-loss variants and lack of control of EGFR wild-type positive clones limit the usefulness of this approach. Chimeric antigen receptor (CAR)-engineered natural killer (NK) cells may represent an alternative immunotherapeutic strategy. For targeting to GBM, we generated variants of the clinically applicable human NK cell line NK-92 that express CARs carrying a composite CD28-CD3ζ domain for signaling, and scFv antibody fragments for cell binding either recognizing EGFR, EGFRvIII, or an epitope common to both antigens. In vitro analysis revealed high and specific cytotoxicity of EGFR-targeted NK-92 against established and primary human GBM cells, which was dependent on EGFR expression and CAR signaling. EGFRvIII-targeted NK-92 only lysed EGFRvIII-positive GBM cells, while dual-specific NK cells expressing a cetuximab-based CAR were active against both types of tumor cells. In immunodeficient mice carrying intracranial GBM xenografts either expressing EGFR, EGFRvIII or both receptors, local treatment with dual-specific NK cells was superior to treatment with the corresponding monospecific CAR NK cells. This resulted in a marked extension of survival without inducing rapid immune escape as observed upon therapy with monospecific effectors. Our results demonstrate that dual targeting of CAR NK cells reduces the risk of immune escape and suggest that EGFR/EGFRvIII-targeted dual-specific CAR NK cells may have potential for adoptive immunotherapy of glioblastoma.

Selection of tumor antigens as targets for immune attack using immunohistochemistry: protein antigens.

PubMed

Zhang, S; Zhang, H S; Cordon-Cardo, C; Ragupathi, G; Livingston, P O

1998-11-01

The relative expression of mucin antigens MUC1, MUC2, MUC3, MUC4, MUC5AC, MUC5B, and MUC7 and glycoprotein antigens KSA, carcinoembryonic antigen, prostate-specific membrane antigen (PSMA), HER-2/neu, and human chorionic gonadotropin-beta on different cancers and normal tissues is difficult to determine from available reports. We have compared the distribution of these antigens by immunohistology on a broad range of malignant and normal tissues. MUC1 expression was most intense in cancers of breast, lung, ovarian, and endometrial origin; MUC2 was most intense in cancers of colon and prostate origin; and MUC5AC was most intense in cancers of breast and gastric origin. MUC4 was intensely expressed in 50% of cancers of colon and pancreas origin, and MUC3, MUC5B, and MUC7 were expressed in a variety of epithelial cancers, but not so intensely. KSA was intensely and uniformly expressed on all epithelial cancers; carcinoembryonic antigen was expressed in most cancers of breast, lung, colon, pancreas, and gastric origin; and PSMA was expressed only in cancers of prostate origin. Human chorionic gonadotropin-beta was expressed on the majority of sarcomas and cancers of breast, lung, and pancreas origin, although intense staining was not seen. Staining on normal tissues was restricted to one or many normal epithelial tissues ranging from MUC3, MUC4, and PSMA, which were expressed only on epithelia of pancreas, stomach, and prostate origin, respectively, to MUC1 and KSA, which were expressed on most normal epithelia. Expression was restricted to the secretory borders of these epithelia while stroma and other normal tissues were completely negative. These results plus the results of the two previous papers (S. Zhang et al, Int. J. Cancer, 73: 42-49, 1997; S. Zhang et al., Int. J. Cancer, 73: 50-56, 1997) in this series provide the basis for selection of multiple cell surface antigens as targets for antibody-mediated attack against these cancers.

A viral deubiquitylating enzyme targets viral RNA-dependent RNA polymerase and affects viral infectivity

PubMed Central

Chenon, Mélanie; Camborde, Laurent; Cheminant, Soizic; Jupin, Isabelle

2012-01-01

Selective protein degradation via the ubiquitin-proteasome system (UPS) plays an essential role in many major cellular processes, including host–pathogen interactions. We previously reported that the tightly regulated viral RNA-dependent RNA polymerase (RdRp) of the positive-strand RNA virus Turnip yellow mosaic virus (TYMV) is degraded by the UPS in infected cells, a process that affects viral infectivity. Here, we show that the TYMV 98K replication protein can counteract this degradation process thanks to its proteinase domain. In-vitro assays revealed that the recombinant proteinase domain is a functional ovarian tumour (OTU)-like deubiquitylating enzyme (DUB), as is the 98K produced during viral infection. We also demonstrate that 98K mediates in-vivo deubiquitylation of TYMV RdRp protein—its binding partner within replication complexes—leading to its stabilization. Finally, we show that this DUB activity contributes to viral infectivity in plant cells. The identification of viral RdRp as a specific substrate of the viral DUB enzyme thus reveals the intricate interplay between ubiquitylation, deubiquitylation and the interaction between viral proteins in controlling levels of RdRp and viral infectivity. PMID:22117220

Expression and characterization of highly antigenic domains of chicken anemia virus viral VP2 and VP3 subunit proteins in a recombinant E. coli for sero-diagnostic applications

PubMed Central

2013-01-01

Background Chicken anemia virus (CAV) is an important viral pathogen that causes anemia and severe immunodeficiency syndrome in chickens worldwide. Generally, CAV infection occurs via vertical transmission in young chicks that are less than two weeks old, which are very susceptible to the disease. Therefore, epidemiological investigations of CAV infection and/or the evaluation of the immunization status of chickens is necessary for disease control. Up to the present, systematically assessing viral protein antigenicity and/or determining the immunorelevant domain(s) of viral proteins during serological testing for CAV infection has never been performed. The expression, production and antigenic characterization of CAV viral proteins such as VP1, VP2 and VP3, and their use in the development of diagnostic kit would be useful for CAV infection prevention. Results Three CAV viral proteins VP1, VP2 and VP3 was separately cloned and expressed in recombinant E. coli. The purified recombinant CAV VP1, VP2 and VP3 proteins were then used as antigens in order to evaluate their reactivity against chicken sera using indirect ELISA. The results indicated that VP2 and VP3 show good immunoreactivity with CAV-positive chicken sera, whereas VP1 was found to show less immunoreactivity than VP2 and VP3. To carry out the further antigenic characterization of the immunorelevant domains of the VP2 and VP3 proteins, five recombinant VP2 subunit proteins (VP2-435N, VP2-396N, VP2-345N, VP2-171C and VP2-318C) and three recombinant VP3 subunit proteins (VP3-123N, VP3-246M, VP3-366C), spanning the defined regions of VP2 and VP3 were separately produced by an E. coli expression system. These peptides were then used as antigens in indirect ELISAs against chicken sera. The results of these ELISAs using truncated recombinant VP2 and VP3 subunit proteins as coating antigen showed that VP2-345N, VP2-396N and VP3-246M gave good immunoreactivity with CAV-positive chicken sera compared to the other

Antigenic variability: Obstacles on the road to vaccines against traditionally difficult targets.

PubMed

Servín-Blanco, R; Zamora-Alvarado, R; Gevorkian, G; Manoutcharian, K

2016-10-02

Despite the impressive impact of vaccines on public health, the success of vaccines targeting many important pathogens and cancers has to date been limited. The burden of infectious diseases today is mainly caused by antigenically variable pathogens (AVPs), which escape immune responses induced by prior infection or vaccination through changes in molecular structures recognized by antibodies or T cells. Extensive genetic and antigenic variability is the major obstacle for the development of new or improved vaccines against "difficult" targets. Alternative, qualitatively new approaches leading to the generation of disease- and patient-specific vaccine immunogens that incorporate complex permanently changing epitope landscapes of intended targets accompanied by appropriate immunomodulators are urgently needed. In this review, we highlight some of the most critical common issues related to the development of vaccines against many pathogens and cancers that escape protective immune responses owing to antigenic variation, and discuss recent efforts to overcome the obstacles by applying alternative approaches for the rational design of new types of immunogens.

Latency reversal and viral clearance to cure HIV-1.

PubMed

Margolis, David M; Garcia, J Victor; Hazuda, Daria J; Haynes, Barton F

2016-07-22

Research toward a cure for human immunodeficiency virus type 1 (HIV-1) infection has joined prevention and treatment efforts in the global public health agenda. A major approach to HIV eradication envisions antiretroviral suppression, paired with targeted therapies to enforce the expression of viral antigen from quiescent HIV-1 genomes, and immunotherapies to clear latent infection. These strategies are targeted to lead to viral eradication--a cure for AIDS. Paired testing of latency reversal and clearance strategies has begun, but additional obstacles to HIV eradication may emerge. Nevertheless, there is reason for optimism that advances in long-acting antiretroviral therapy and HIV prevention strategies will contribute to efforts in HIV cure research and that the implementation of these efforts will synergize to markedly blunt the effect of the HIV pandemic on society. Copyright © 2016, American Association for the Advancement of Science.

KSHV Targeted Therapy: An Update on Inhibitors of Viral Lytic Replication

PubMed Central

Coen, Natacha; Duraffour, Sophie; Snoeck, Robert; Andrei, Graciela

2014-01-01

Kaposi’s sarcoma-associated herpesvirus (KSHV) is the causative agent of Kaposi’s sarcoma, primary effusion lymphoma and multicentric Castleman’s disease. Since the discovery of KSHV 20 years ago, there is still no standard treatment and the management of virus-associated malignancies remains toxic and incompletely efficacious. As the majority of tumor cells are latently infected with KSHV, currently marketed antivirals that target the virus lytic cycle have shown inconsistent results in clinic. Nevertheless, lytic replication plays a major role in disease progression and virus dissemination. Case reports and retrospective studies have pointed out the benefit of antiviral therapy in the treatment and prevention of KSHV-associated diseases. As a consequence, potent and selective antivirals are needed. This review focuses on the anti-KSHV activity, mode of action and current status of antiviral drugs targeting KSHV lytic cycle. Among these drugs, different subclasses of viral DNA polymerase inhibitors and compounds that do not target the viral DNA polymerase are being discussed. We also cover molecules that target cellular kinases, as well as the potential of new drug targets and animal models for antiviral testing. PMID:25421895

Pseudorabies virus glycoprotein gIII is a major target antigen for murine and swine virus-specific cytotoxic T lymphocytes.

PubMed Central

Zuckermann, F A; Zsak, L; Mettenleiter, T C; Ben-Porat, T

1990-01-01

Pseudorabies virus (PrV) is the etiological agent of Aujeszky's disease, a disease that causes heavy economic losses in the swine industry. A rational approach to the generation of an effective vaccine against this virus requires an understanding of the immune response induced by it and of the role of the various viral antigens in inducing such a response. We have constructed mutants of PrV [strain PrV (Ka)] that differ from each other only in expression of the viral nonessential glycoproteins gI, gp63, gX, and gIII (i.e., are otherwise isogenic). These mutants were used to ascertain the importance of each of the nonessential glycoproteins in eliciting a PrV-specific cytotoxic T-lymphocyte (CTL) response in mice and pigs. Immunization of DBA/2 mice and pigs with a thymidine kinase-deficient (TK-) mutant of PrV elicits the formation of cytotoxic cells that specifically lyse syngeneic infected target cells. These PrV-specific cytolytic cells have the phenotype of major histocompatibility complex class I antigen-restricted CTLs. The relative number of CTLs specific for glycoproteins gI, gp63, gX, and gIII induced in mice vaccinated with a TK- mutant of PrV was ascertained by comparing their levels of cytotoxicity against syngeneic cells infected with either wild-type virus or gI-/gp63-, gX-, or gIII- virus deletion mutants. The PrV-specific CLTs were significantly less effective in lysing gIII(-)-infected targets than in lysing gI-/gp63-, gX-, or wild-type-infected targets. The in vitro secondary CTL response of lymphocytes obtained from either mice or pigs 6 or more weeks after immunization with a TK- mutant of PrV was also tested. Lymphocytes obtained from these animals were cultured with different glycoprotein-deficient mutants of PrV, and their cytolytic activities against wild-type-infected targets were ascertained. The importance of each of the nonessential viral glycoproteins in eliciting CTLs was assessed from the effectiveness of each of the virus mutants to

Ubiquitinated Proteins Isolated From Tumor Cells Are Efficient Substrates for Antigen Cross-Presentation.

PubMed

Yu, Guangjie; Moudgil, Tarsem; Cui, Zhihua; Mou, Yongbin; Wang, Lixin; Fox, Bernard A; Hu, Hong-Ming

2017-06-01

We have previously shown that inhibition of the proteasome causes defective ribosomal products to be shunted into autophagosomes and subsequently released from tumor cells as defective ribosomal products in Blebs (DRibbles). These DRibbles serve as an excellent source of antigens for cross-priming of tumor-specific T cells. Here, we examine the role of ubiquitinated proteins (Ub-proteins) in this pathway. Using purified Ub-proteins from tumor cells that express endogenous tumor-associated antigen or exogenous viral antigen, we tested the ability of these proteins to stimulate antigen-specific T-cell responses, by activation of monocyte-derived dendritic cells generated from human peripheral blood mononuclear cells. Compared with total cell lysates, we found that purified Ub-proteins from both a gp100-specific melanoma cell line and from a lung cancer cell line expressing cytomegalovirus pp65 antigen produced a significantly higher level of IFN-γ in gp100- or pp65-specific T cells, respectively. In addition, Ub-proteins from an allogeneic tumor cell line could be used to stimulate tumor-infiltrating lymphocytes isolated and expanded from non-small cell lung cancer patients. These results establish that Ub-proteins provide a relevant source of antigens for cross-priming of antitumor immune responses in a variety of settings, including endogenous melanoma and exogenous viral antigen presentation, as well as antigen-specific tumor-infiltrating lymphocytes. Thus, ubiquitin can be used as an affinity tag to enrich for unknown tumor-specific antigens from tumor cell lysates to stimulate tumor-specific T cells ex vivo or to be used as vaccines to target short-lived proteins.

Rabies virus glycoprotein as a carrier for anthrax protective antigen

DOE Office of Scientific and Technical Information (OSTI.GOV)

Smith, Mary Ellen; Koser, Martin; Xiao Sa

2006-09-30

Live viral vectors expressing foreign antigens have shown great promise as vaccines against viral diseases. However, safety concerns remain a major problem regarding the use of even highly attenuated viral vectors. Using the rabies virus (RV) envelope protein as a carrier molecule, we show here that inactivated RV particles can be utilized to present Bacillus anthracis protective antigen (PA) domain-4 in the viral membrane. In addition to the RV glycoprotein (G) transmembrane and cytoplasmic domains, a portion of the RV G ectodomain was required to express the chimeric RV G anthrax PA on the cell surface. The novel antigen wasmore » also efficiently incorporated into RV virions. Mice immunized with the inactivated recombinant RV virions exhibited seroconversion against both RV G and anthrax PA, and a second inoculation greatly increased these responses. These data demonstrate that a viral envelope protein can carry a bacterial protein and that a viral carrier can display whole polypeptides compared to the limited epitope presentation of previous viral systems.« less

Viral interference with DNA repair by targeting of the single-stranded DNA binding protein RPA.

PubMed

Banerjee, Pubali; DeJesus, Rowena; Gjoerup, Ole; Schaffhausen, Brian S

2013-10-01

Correct repair of damaged DNA is critical for genomic integrity. Deficiencies in DNA repair are linked with human cancer. Here we report a novel mechanism by which a virus manipulates DNA damage responses. Infection with murine polyomavirus sensitizes cells to DNA damage by UV and etoposide. Polyomavirus large T antigen (LT) alone is sufficient to sensitize cells 100 fold to UV and other kinds of DNA damage. This results in activated stress responses and apoptosis. Genetic analysis shows that LT sensitizes via the binding of its origin-binding domain (OBD) to the single-stranded DNA binding protein replication protein A (RPA). Overexpression of RPA protects cells expressing OBD from damage, and knockdown of RPA mimics the LT phenotype. LT prevents recruitment of RPA to nuclear foci after DNA damage. This leads to failure to recruit repair proteins such as Rad51 or Rad9, explaining why LT prevents repair of double strand DNA breaks by homologous recombination. A targeted intervention directed at RPA based on this viral mechanism could be useful in circumventing the resistance of cancer cells to therapy.

Nonviral RNA transfection to transiently modify T cells with chimeric antigen receptors for adoptive therapy.

PubMed

Riet, Tobias; Holzinger, Astrid; Dörrie, Jan; Schaft, Niels; Schuler, Gerold; Abken, Hinrich

2013-01-01

Redirecting T cells with a chimeric antigen receptor (CAR) of predefined specificity showed remarkable efficacy in the adoptive therapy trials of malignant diseases. The CAR consists of a single chain fragment of variable region (scFv) antibody targeting domain covalently linked to the CD3ζ signalling domain of the T cell receptor complex to mediate T cell activation upon antigen engagement. By using an antibody-derived targeting domain a CAR can potentially redirect T cells towards any target expressed on the cell surface as long as a binding domain is available. Antibody-mediated targeting moreover circumvents MHC restriction of the targeted antigen, thereby broadening the potential of applicability of adoptive T cell therapy. While T cells were so far genetically modified by viral transduction, transient modification with a CAR by RNA transfection gained increasing interest during the last years. This chapter focuses on methods to modify human T cells from peripheral blood with a CAR by electroporation of in vitro transcribed RNA and to test modified T cells for function for use in adoptive immunotherapy.

Detection of hepatitis A viral antigen by radioimmunoassay

DOE Office of Scientific and Technical Information (OSTI.GOV)

Hollinger, F.B.; Bradley, D.W.; Maynard, J.E.

1975-11-01

With coded samples, the effectiveness and specificity of a micro-SPIRA procedure for rapidly and quantitatively detecting type A hepatitis-associated antigen in large numbers of specimens from infected liver, stool, or serum has been demonstrated. Samples which were judged to be negative by IEM were found to contain significant levels of HAV antigen by this immunoradiometric technique. The detection of significant levels of HAV antigen in infected chimpanzees supports epidemiologic evidence of viremia during the acute stage of the disease. The results of this study suggest that the diagnosis of type A hepatitis by a convention serologic procedure may now bemore » at hand. (auth)« less

Carbamylated albumin is one of the target antigens of anti-carbamylated protein antibodies.

PubMed

Nakabo, Shuichiro; Hashimoto, Motomu; Ito, Shinji; Furu, Moritoshi; Ito, Hiromu; Fujii, Takao; Yoshifuji, Hajime; Imura, Yoshitaka; Nakashima, Ran; Murakami, Kosaku; Kuramoto, Nobuo; Tanaka, Masao; Satoh, Junko; Ishigami, Akihito; Morita, Satoshi; Mimori, Tsuneyo; Ohmura, Koichiro

2017-07-01

Anti-carbamylated protein (anti-CarP) antibodies are detected in RA patients. Fetal calf serum is used as an antigen source in anti-CarP ELISA, and the precise target antigens have not been found. We aimed to identify the target antigens of anti-CarP antibodies. Western blotting of anti-CarP antibodies was conducted. Anti-carbamylated human albumin (CarALB) antibody was detected by in-house ELISA for 493 RA patients and 144 healthy controls (HCs). An inhibition ELISA of anti-CarP antibodies by CarALB and citrullinated albumin (citALB) was performed using eight RA patients' sera. Serum CarALB was detected by liquid chromatography-tandem mass spectroscopy (LC/MS/MS), and the serum MPO concentration was measured by ELISA. We focused on carbamylated albumin because it corresponded to the size of the thickest band detected by western blotting of anti-CarP antibodies. Anti-CarALB antibody was detected in 31.4% of RA patients, and the correlation of the titres between anti-CarALB and anti-CarP was much closer than that between anti-citALB and anti-CCP antibodies (ρ = 0.59 and ρ = 0.16, respectively). The inhibition ELISA showed that anti-CarP antibodies were inhibited by CarALB, but not by citALB. CarALB was detected in sera from RA patients by LC/MS/MS. The serum MPO concentration was correlated with disease activity and was higher in RA patients with anti-CarALB antibody than in those without. We found that carbamylated albumin is a novel target antigen of anti-CarP antibodies, and it is the first reported target antigen that has not been reported as the target of ACPA. © The Author 2017. Published by Oxford University Press on behalf of the British Society for Rheumatology. All rights reserved. For Permissions, please email: journals.permissions@oup.com

Polymer blend particles with defined compositions for targeting antigen to both class I and II antigen presentation pathways

PubMed Central

Tran, Kenny K.; Zhan, Xi; Shen, Hong

2013-01-01

Defense against many persistent and difficult-to-treat diseases requires a combination of humoral, CD4+ and CD8+ T cell responses, which necessitates targeting antigens to both class I and II antigen presentation pathways. In this study, we developed polymer blend particles by mixing two functionally unique polymers, poly(lactide-co-glycolide) (PLGA) and a pH-responsive polymer, poly(dimethylaminoethyl methacrylate-co-propylacrylic acid-co-butyl methacrylate) (DMAEMA-co-PAA-co-BMA). We showed polymer blend particles enabled the delivery of antigens into both class I and II antigen presentation pathways in vitro. Increasing the ratio of the pH-responsive polymer in blend particles increased the degree of class I antigen presentation, while maintaining high levels of class II antigen presentation. In a mouse model, we demonstrated that a significantly higher and sustained level of CD4+ and CD8+ T cell responses, and comparable antibody responses, were elicited with polymer blend particles than PLGA particles and a conventional vaccine, Alum. The polymer blend particles offer a potential vaccine delivery platform to generate a combination of humoral and cell-mediated immune responses that insure robust and long-lasting immunity against many infectious diseases and cancers. PMID:24124123

Targeting of plant-derived vaccine antigens to immunoresponsive mucosal sites.

PubMed

Rigano, M Manuela; Sala, Francesco; Arntzen, Charles J; Walmsley, Amanda M

2003-01-30

Most pathogenic microorganisms enter their host via the mucosal surfaces lining the digestive, respiratory and urino-reproductive tracts of the body. The most efficient means of protecting these surfaces is through mucosal immunization. Transgenic plants are safe and inexpensive vehicles to produce and mucosally deliver protective antigens. However, the application of this technology is limited by the poor response of the immune system to non-particulate, subunit vaccines. Co-delivery of therapeutic proteins with targeting proteins, such as the B subunit of the Escherichia coli heat labile enterotoxin (LTB), could increase the effectiveness of such antigens.

Asymmetric Assembly of Merkel Cell Polyomavirus Large T-Antigen Origin Binding Domains at the Viral Origin

DOE Office of Scientific and Technical Information (OSTI.GOV)

C Harrison; G Meinke; H Kwun

2011-12-31

The double-stranded DNA polyomavirus Merkel cell polyomavirus (MCV) causes Merkel cell carcinoma, an aggressive but rare human skin cancer that most often affects immunosuppressed and elderly persons. As in other polyomaviruses, the large T-antigen of MCV recognizes the viral origin of replication by binding repeating G(A/G)GGC pentamers. The spacing, number, orientation, and necessity of repeats for viral replication differ, however, from other family members such as SV40 and murine polyomavirus. We report here the 2.9 {angstrom} crystal structure of the MCV large T-antigen origin binding domain (OBD) in complex with a DNA fragment from the MCV origin of replication. Consistentmore » with replication data showing that three of the G(A/G)GGC-like binding sites near the center of the origin are required for replication, the crystal structure contains three copies of the OBD. This stoichiometry was verified using isothermal titration calorimetry. The affinity for G(A/G)GGC-containing double-stranded DNA was found to be {approx} 740 nM, approximately 8-fold weaker than the equivalent domain in SV40 for the analogous region of the SV40 origin. The difference in affinity is partially attributable to DNA-binding residue Lys331 (Arg154 in SV40). In contrast to SV40, a small protein-protein interface is observed between MCV OBDs when bound to the central region of the origin. This protein-protein interface is reminiscent of that seen in bovine papilloma virus E1 protein. Mutational analysis indicates, however, that this interface contributes little to DNA binding energy.« less

Molecular Targeted Viral Nanoparticles as Tools for Imaging Cancer

PubMed Central

Cho, C.F.; Sourabh, S.; Simpson, E.J.; Steinmetz, N.F.; Luyt, L.G.; Lewis, J.D.

2015-01-01

Viral nanoparticles (VNPs) are a novel class of bionanomaterials that harness the natural biocompatibility of viruses for the development of therapeutics, vaccines, and imaging tools. The plant virus, cowpea mosaic virus (CPMV), has been successfully engineered to create novel cancer-targeted imaging agents by incorporating fluorescent dyes, polyethylene glycol (PEG) polymers, and targeting moieties. Using straightforward conjugation strategies, VNPs with high selectivity for cancer-specific molecular targets can be synthesized for in vivo imaging of tumors. Here we describe the synthesis and purification of CPMV-based VNPs, the functionalization of these VNPs using click chemistry, and their use for imaging xenograft tumors in animal models. VNPs decorated with fluorescent dyes, PEG, and targeting ligands can be synthesized in one day, and imaging studies can be performed over hours, days, or weeks, depending on the application. PMID:24243252

Dendritic cell–targeted lentiviral vector immunization uses pseudotransduction and DNA-mediated STING and cGAS activation

PubMed Central

Kim, Jocelyn T.; Liu, Yarong; Kulkarni, Rajan P.; Lee, Kevin K.; Dai, Bingbing; Lovely, Geoffrey; Ouyang, Yong; Wang, Pin; Yang, Lili; Baltimore, David

2018-01-01

Dendritic cell (DC) activation and antigen presentation are critical for efficient priming of T cell responses. Here, we study how lentiviral vectors (LVs) deliver antigen and activate DCs to generate T cell immunization in vivo. We report that antigenic proteins delivered in vector particles via pseudotransduction were sufficient to stimulate an antigen-specific immune response. The delivery of the viral genome encoding the antigen increased the magnitude of this response in vivo but was irrelevant in vitro. Activation of DCs by LVs was independent of MyD88, TRIF, and MAVS, ruling out an involvement of Toll-like receptor or RIG-I–like receptor signaling. Cellular DNA packaged in LV preparations induced DC activation by the host STING (stimulator of interferon genes) and cGAS (cyclic guanosine monophosphate–adenosine monophosphate synthase) pathway. Envelope-mediated viral fusion also activated DCs in a phosphoinositide 3-kinase–dependent but STING-independent process. Pseudotransduction, transduction, viral fusion, and delivery of cellular DNA collaborate to make the DC-targeted LV preparation an effective immunogen. PMID:28733470

Antigen discovery and delivery of subunit vaccines by nonliving bacterial ghost vectors.

PubMed

Walcher, Petra; Mayr, Ulrike B; Azimpour-Tabrizi, Chakameh; Eko, Francis O; Jechlinger, Wolfgang; Mayrhofer, Peter; Alefantis, Tim; Mujer, Cesar V; DelVecchio, Vito G; Lubitz, Werner

2004-12-01

The bacterial ghost (BG) platform system is a novel vaccine delivery system endowed with intrinsic adjuvant properties. BGs are nonliving Gram-negative bacterial cell envelopes which are devoid of their cytoplasmic contents, yet maintain their cellular morphology and antigenic structures, including bioadhesive properties. The main advantages of BGs as carriers of subunit vaccines include their ability to stimulate a high immune response and to target the carrier itself to primary antigen-presenting cells. The intrinsic adjuvant properties of BGs enhance the immune response to target antigens, including T-cell activation and mucosal immunity. Since native and foreign antigens can be carried in the envelope complex of BGs, combination vaccines with multiple antigens of diverse origin can be presented to the immune system simultaneously. Beside the capacity of BGs to function as carriers of protein antigens, they also have a high loading capacity for DNA. Thus, loading BGs with recombinant DNA takes advantage of the excellent bioavailability for DNA-based vaccines and the high expression rates of the DNA-encoded antigens in target cell types such as macrophages and dendritic cells. There are many spaces within BGs including the inner and outer membranes, the periplasmic space and the internal lumen which can carry antigens, DNA or mediators of the immune response. All can be used for subunit antigen to design new vaccine candidates with particle presentation technology. In addition, the fact that BGs can also carry piggyback large-size foreign antigen particles, increases the technologic usefulness of BGs as combination vaccines against viral and bacterial pathogens. Furthermore, the BG antigen carriers can be stored as freeze-dried preparations at room temperature for extended periods without loss of efficacy. The potency, safety and relatively low production cost of BGs offer a significant technical advantage over currently utilized vaccine technologies.

Kinetics of antibody-induced modulation of respiratory syncytial virus antigens in a human epithelial cell line

PubMed Central

Sarmiento, Rosa E; Tirado, Rocio G; Valverde, Laura E; Gómez-Garcia, Beatriz

2007-01-01

Background The binding of viral-specific antibodies to cell-surface antigens usually results in down modulation of the antigen through redistribution of antigens into patches that subsequently may be internalized by endocytosis or may form caps that can be expelled to the extracellular space. Here, by use of confocal-laser-scanning microscopy we investigated the kinetics of the modulation of respiratory syncytial virus (RSV) antigen by RSV-specific IgG. RSV-infected human epithelial cells (HEp-2) were incubated with anti-RSV polyclonal IgG and, at various incubation times, the RSV-cell-surface-antigen-antibody complexes (RSV Ag-Abs) and intracellular viral proteins were detected by indirect immunoflourescence. Results Interaction of anti-RSV polyclonal IgG with RSV HEp-2 infected cells induced relocalization and aggregation of viral glycoproteins in the plasma membrane formed patches that subsequently produced caps or were internalized through clathrin-mediated endocytosis participation. Moreover, the concentration of cell surface RSV Ag-Abs and intracellular viral proteins showed a time dependent cyclic variation and that anti-RSV IgG protected HEp-2 cells from viral-induced death. Conclusion The results from this study indicate that interaction between RSV cell surface proteins and specific viral antibodies alter the expression of viral antigens expressed on the cells surface and intracellular viral proteins; furthermore, interfere with viral induced destruction of the cell. PMID:17608950

Bovine single chain Fv antibody inhibits bovine herpesvirus-1 infectivity by targeting viral glycoprotein D.

PubMed

Xu, Jian; Wu, Jing; Jiang, Bo; He, Houjun; Zhang, Xixi; Li, Xiaoyang; Yang, Dawei; Huang, Xiufen; Sealy, Joshua E; Iqbal, Munir; Li, Yongqing

2017-12-01

Glycoprotein D (gD) of bovine herpesvirus-1 (BoHV-1) is essential for attachment and penetration of cells during infection and is a major target for neutralizing antibodies during an adaptive immune response. Currently there are no recombinant antibodies capable of binding gD epitopes for use in treating BoHV-1 infection. In this study, a bovine scFv gene derived from a hybridoma secreting monoclonal antibodies (McAbs) against the amino acid motif MEESKGYEPP of gD was expressed in E. coli. Molecular modeling, western blot and ELISA analysis showed that this scFv had a high affinity for BoHV-1 gD, with a Kd of 161.2 ± 37.58 nM and for whole BoHV-1 virus, with a Kd of 67.44 ± 16.99 nM. In addition, this scFv displayed a high affinity for BoHV-1 antigen in an ELISA and competed with BoHV-1 anti-serum in a competitive ELISA. Immunofluorescence assay (IFA) and laser confocal microscopy showed that this scFv could efficiently bind to and be internalized by BoHV-1 infected Madin-Darby bovine kidney (MDBK) cells. Importantly, this scFv was shown to inhibit BoHV-1 infectivity and to reduce the number of viral plaques by blocking viral attachment to MDBK cells. Our study suggests that this bovine single-chain antibody could be developed for use as a diagnostic and therapeutic agent against BoHV-1 infection in cattle.

[Difference in target antigens between central tolerance and peripheral tolerance deficiencies].

PubMed

Chida, Natsuko; Kobayashi, Ichiro

2015-01-01

Failure of the immunotolerance mechanisms causes multiple organ-specific autoimmune disorders. Mutations of autoimmune regulator (AIRE) gene result in central immunotolerance deficiency named autoimmune polyendocrinopathy, candidiasis, ectodermal dystrophy (APECED). Mutations of FOXP3 genes cause regulatory T cell (Treg) deficiency named immune dysregulation, polyendocrinopathy, enteropathy, X-linked (IPEX) syndrome. Because T cell tolerance influences B cell tolerance, autoantibodies seem to reflect the presence of autoreactive T cells with the same antigen specificity. To date many differences in both clinical features and autoantibody profiles have been described between APECED and IPEX syndrome. In addition to the differences in target organs, we have found differences in the target antigens in the same organ, small intestine, between both disorders; anti-autoimmune enteropathy-related 75 kDa antigen (AIE-75) antibodies are specific to IPEX syndrome, whereas anti-tryptophan hydroxylase-1 (TPH-1) antibodies are specific to APECED. These facts suggest that immunotolerance to AIE-75 depends on the Treg, whereas the tolerance to TPH-1 depends on the central mechanisms. Furthermore, given the earlier onset and more serious clinical features of IPEX syndrome than APECED, physiological roles of Aire on the selection of Treg may be, if present, limited.

Premature activation of the paramyxovirus fusion protein before target cell attachment with corruption of the viral fusion machinery.

PubMed

Farzan, Shohreh F; Palermo, Laura M; Yokoyama, Christine C; Orefice, Gianmarco; Fornabaio, Micaela; Sarkar, Aurijit; Kellogg, Glen E; Greengard, Olga; Porotto, Matteo; Moscona, Anne

2011-11-04

Paramyxoviruses, including the childhood pathogen human parainfluenza virus type 3, enter host cells by fusion of the viral and target cell membranes. This fusion results from the concerted action of its two envelope glycoproteins, the hemagglutinin-neuraminidase (HN) and the fusion protein (F). The receptor-bound HN triggers F to undergo conformational changes that render it competent to mediate fusion of the viral and cellular membranes. We proposed that, if the fusion process could be activated prematurely before the virion reaches the target host cell, infection could be prevented. We identified a small molecule that inhibits paramyxovirus entry into target cells and prevents infection. We show here that this compound works by an interaction with HN that results in F-activation prior to receptor binding. The fusion process is thereby prematurely activated, preventing fusion of the viral membrane with target cells and precluding viral entry. This first evidence that activation of a paramyxovirus F can be specifically induced before the virus contacts its target cell suggests a new strategy with broad implications for the design of antiviral agents.

TNF-induced target cell killing by CTL activated through cross-presentation.

PubMed

Wohlleber, Dirk; Kashkar, Hamid; Gärtner, Katja; Frings, Marianne K; Odenthal, Margarete; Hegenbarth, Silke; Börner, Carolin; Arnold, Bernd; Hämmerling, Günter; Nieswandt, Bernd; van Rooijen, Nico; Limmer, Andreas; Cederbrant, Karin; Heikenwalder, Mathias; Pasparakis, Manolis; Protzer, Ulrike; Dienes, Hans-Peter; Kurts, Christian; Krönke, Martin; Knolle, Percy A

2012-09-27

Viruses can escape cytotoxic T cell (CTL) immunity by avoiding presentation of viral components via endogenous MHC class I antigen presentation in infected cells. Cross-priming of viral antigens circumvents such immune escape by allowing noninfected dendritic cells to activate virus-specific CTLs, but they remain ineffective against infected cells in which immune escape is functional. Here, we show that cross-presentation of antigen released from adenovirus-infected hepatocytes by liver sinusoidal endothelial cells stimulated cross-primed effector CTLs to release tumor necrosis factor (TNF), which killed virus-infected hepatocytes through caspase activation. TNF receptor signaling specifically eliminated infected hepatocytes that showed impaired anti-apoptotic defense. Thus, CTL immune surveillance against infection relies on two similarly important but distinct effector functions that are both MHC restricted, requiring either direct antigen recognition on target cells and canonical CTL effector function or cross-presentation and a noncanonical effector function mediated by TNF. Copyright © 2012 The Authors. Published by Elsevier Inc. All rights reserved.

Heterobivalent Imaging Agents for Simultaneous Targeting Prostate-Specific Membrane Antigen (PSMA) and Hepsin

DTIC Science & Technology

2014-11-01

Prostate-Specific Membrane Antigen ( PSMA ) and Hepsin PRINCIPAL INVESTIGATOR: Youngjoo Byun, Ph. D. CONTRACTING ORGANIZATION: Korea...Simultaneous Targeting Prostate-Specific Membrane Antigen ( PSMA ) and Hepsin 5b. GRANT NUMBER W81XWH-10-1-0189 5c. PROGRAM ELEMENT NUMBER 6...heterobivalent conjugates of PSMA /hepsin-binding ligands labeled with optical dyes or radionuclides. The sensitivity and accuracy of prostate cancer

Multivalent display of proteins on viral nanoparticles using molecular recognition and chemical ligation strategies

PubMed Central

Venter, P. Arno; Dirksen, Anouk; Thomas, Diane; Manchester, Marianne; Dawson, Philip E.; Schneemann, Anette

2011-01-01

Multivalent display of heterologous proteins on viral nanoparticles forms a basis for numerous applications in nanotechnology, including vaccine development, targeted therapeutic delivery and tissue-specific bio-imaging. In many instances, precise placement of proteins is required for optimal functioning of the supramolecular assemblies, but orientation- and site-specific coupling of proteins to viral scaffolds remains a significant technical challenge. We have developed two strategies that allow for controlled attachment of a variety of proteins on viral particles using covalent and noncovalent principles. In one strategy, an interaction between domain 4 of anthrax protective antigen and its receptor was used to display multiple copies of a target protein on virus-like particles. In the other, expressed protein ligation and aniline-catalyzed oximation was used to covalently display a model protein. The latter strategy, in particular, yielded nanoparticles that induced potent immune responses to the coupled protein, suggesting potential applications in vaccine development. PMID:21545187

New targets for immunotherapy-based treatment of HPV-related cancers | Center for Cancer Research

Cancer.gov

Scientists at the Center for Cancer Research and three other cancer research institutions show that immunotherapy treatments that resulted in complete regression of metastatic cervical cancer largely targeted two non-viral antigens. Read more…  

Identification of influenza A nucleoprotein body domain residues essential for viral RNA expression expose antiviral target.

PubMed

Davis, Alicia M; Ramirez, Jose; Newcomb, Laura L

2017-02-07

Influenza A virus is controlled with yearly vaccination while emerging global pandemics are kept at bay with antiviral medications. Unfortunately, influenza A viruses have emerged resistance to approved influenza antivirals. Accordingly, there is an urgent need for novel antivirals to combat emerging influenza A viruses resistant to current treatments. Conserved viral proteins are ideal targets because conserved protein domains are present in most, if not all, influenza subtypes, and are presumed less prone to evolve viable resistant versions. The threat of an antiviral resistant influenza pandemic justifies our study to identify and characterize antiviral targets within influenza proteins that are highly conserved. Influenza A nucleoprotein (NP) is highly conserved and plays essential roles throughout the viral lifecycle, including viral RNA synthesis. Using NP crystal structure, we targeted accessible amino acids for substitution. To characterize the NP proteins, reconstituted viral ribonucleoproteins (vRNPs) were expressed in 293 T cells, RNA was isolated, and reverse transcription - quantitative PCR (RT-qPCR) was employed to assess viral RNA expressed from reconstituted vRNPs. Location was confirmed using cellular fractionation and western blot, along with observation of NP-GFP fusion proteins. Nucleic acid binding, oligomerization, and vRNP formation, were each assessed with native gel electrophoresis. Here we report characterization of an accessible and conserved five amino acid region within the NP body domain that plays a redundant but essential role in viral RNA synthesis. Our data demonstrate substitutions in this domain did not alter NP localization, oligomerization, or ability to bind nucleic acids, yet resulted in a defect in viral RNA expression. To define this region further, single and double amino acid substitutions were constructed and investigated. All NP single substitutions were functional, suggesting redundancy, yet different combinations of

Viral RNA Intermediates as Targets for Detection and Discovery of Novel and Emerging Mosquito-Borne Viruses

PubMed Central

Yam, Alice Wei Yee; Colmant, Agathe M. G.; McLean, Breeanna J.; Prow, Natalie A.; Watterson, Daniel; Hall-Mendelin, Sonja; Warrilow, David; Ng, Mah-Lee; Khromykh, Alexander A.; Hall, Roy A.

2015-01-01

Mosquito-borne viruses encompass a range of virus families, comprising a number of significant human pathogens (e.g., dengue viruses, West Nile virus, Chikungunya virus). Virulent strains of these viruses are continually evolving and expanding their geographic range, thus rapid and sensitive screening assays are required to detect emerging viruses and monitor their prevalence and spread in mosquito populations. Double-stranded RNA (dsRNA) is produced during the replication of many of these viruses as either an intermediate in RNA replication (e.g., flaviviruses, togaviruses) or the double-stranded RNA genome (e.g., reoviruses). Detection and discovery of novel viruses from field and clinical samples usually relies on recognition of antigens or nucleotide sequences conserved within a virus genus or family. However, due to the wide antigenic and genetic variation within and between viral families, many novel or divergent species can be overlooked by these approaches. We have developed two monoclonal antibodies (mAbs) which show co-localised staining with proteins involved in viral RNA replication in immunofluorescence assay (IFA), suggesting specific reactivity to viral dsRNA. By assessing binding against a panel of synthetic dsRNA molecules, we have shown that these mAbs recognise dsRNA greater than 30 base pairs in length in a sequence-independent manner. IFA and enzyme-linked immunosorbent assay (ELISA) were employed to demonstrate detection of a panel of RNA viruses from several families, in a range of cell types. These mAbs, termed monoclonal antibodies to viral RNA intermediates in cells (MAVRIC), have now been incorporated into a high-throughput, economical ELISA-based screening system for the detection and discovery of viruses from mosquito populations. Our results have demonstrated that this simple system enables the efficient detection and isolation of a range of known and novel viruses in cells inoculated with field-caught mosquito samples, and represents

[Progress in application of targeting viral vector regulated by microRNA in gene therapy: a review].

PubMed

Zhang, Guohai; Wang, Qizhao; Zhang, Jinghong; Xu, Ruian

2010-06-01

A safe and effective targeting viral vector is the key factor for successful clinical gene therapy. microRNA, a class of small, single-stranded endogenous RNAs, act as post-transcriptional regulators of gene expression. The discovery of these kind regulatory elements provides a new approach to regulate gene expression more accurately. In this review, we elucidated the principle of microRNA in regulation of targeting viral vector. The applications of microRNA in the fields of elimination contamination from replication competent virus, reduction of transgene-specific immunity, promotion of cancer-targeted gene therapy and development of live attenuated vaccines were also discussed.

FLT3-regulated antigens as targets for leukemia-reactive cytotoxic T lymphocytes

PubMed Central

Brackertz, B; Conrad, H; Daniel, J; Kast, B; Krönig, H; Busch, D H; Adamski, J; Peschel, C; Bernhard, H

2011-01-01

The FMS-like tyrosine kinase 3 (FLT3) is highly expressed in acute myeloid leukemia (AML). Internal tandem duplications (ITD) of the juxtamembrane domain lead to the constitutive activation of the FLT3 kinase inducing the activation of multiple genes, which may result in the expression of leukemia-associated antigens (LAAs). We analyzed the regulation of LAA in FLT3-wild-type (WT)- and FLT3-ITD+ myeloid cells to identify potential targets for antigen-specific immunotherapy for AML patients. Antigens, such as PR-3, RHAMM, Survivin, WT-1 and PRAME, were upregulated by constitutively active FLT3-ITD as well as FLT3-WT activated by FLT3 ligand (FL). Cytotoxic T-cell (CTL) clones against PR-3, RHAMM, Survivin and an AML-directed CTL clone recognized AML cell lines and primary AML blasts expressing FLT3-ITD, as well as FLT3-WT+ myeloid dendritic cells in the presence of FL. Downregulation of FLT3 led to the abolishment of CTL recognition. Comparing our findings concerning LAA upregulation by the FLT3 kinase with those already made for the Bcr-Abl kinase, we found analogies in the LAA expression pattern. Antigens upregulated by both FLT3 and Bcr-Abl may be promising targets for the development of immunotherapeutical approaches against myeloid leukemia of different origin. PMID:22829124

The essential role of guinea pig cytomegalovirus (GPCMV) IE1 and IE2 homologs in viral replication and IE1-mediated ND10 targeting

DOE Office of Scientific and Technical Information (OSTI.GOV)

Hornig, Julia; Choi, K. Yeon; McGregor, Alistair,

Guinea pig cytomegalovirus (GPCMV) immediate early proteins, IE1 and IE2, demonstrated structural and functional homologies with human cytomegalovirus (HCMV). GPCMV IE1 and IE2 co-localized in the nucleus with each other, the viral polymerase and guinea pig ND10 components (gpPML, gpDaxx, gpSp100, gpATRX). IE1 showed direct interaction with ND10 components by immunoprecipitation unlike IE2. Additionally, IE1 protein disrupted ND10 bodies. IE1 mutagenesis mapped the nuclear localization signal to the C-terminus and identified the core domain for gpPML interaction. Individual knockout of GPCMV GP122 or GP123 (IE2 and IE1 unique exons respectively) was lethal to the virus. However, an IE1 mutant (codonsmore » 234–474 deleted), was viable with attenuated viral growth kinetics and increased susceptibility to type I interferon (IFN-I). In HCMV, the IE proteins are important T cell target antigens. Consequently, characterization of the homologs in GPCMV provides a basis for their evaluation in candidate vaccines against congenital infection.« less

The essential role of guinea pig cytomegalovirus (GPCMV) IE1 and IE2 homologs in viral replication and IE1-mediated ND10 targeting

PubMed Central

Hornig, Julia; Choi, K. Yeon; McGregor, Alistair

2017-01-01

Guinea pig cytomegalovirus (GPCMV) immediate early proteins, IE1 and IE2, demonstrated structural and functional homologies with human cytomegalovirus (HCMV). GPCMV IE1 and IE2 co-localized in the nucleus with each other, the viral polymerase and guinea pig ND10 components (gpPML, gpDaxx, gpSp100, gpATRX). IE1 showed direct interaction with ND10 components by immunoprecipitation unlike IE2. Additionally, IE1 protein disrupted ND10 bodies. IE1 mutagenesis mapped the nuclear localization signal to the C-terminus and identified the core domain for gpPML interaction. Individual knockout of GPCMV GP122 or GP123 (IE2 and IE1 unique exons respectively) was lethal to the virus. However, an IE1 mutant (codons 234–474 deleted), was viable with attenuated viral growth kinetics and increased susceptibility to type I interferon (IFN-I). In HCMV, the IE proteins are important T cell target antigens. Consequently, characterization of the homologs in GPCMV provides a basis for their evaluation in candidate vaccines against congenital infection. PMID:28189970

Prostate Stem Cell Antigen: A Prospective Therapeutic and Diagnostic Target

PubMed Central

Raff, Adam B.; Gray, Andrew; Kast, W. Martin

2009-01-01

The development of novel clinical tools to combat cancer is an intense field of research and recent efforts have been directed at the identification of proteins that may provide diagnostic, prognostic and/or therapeutic applications due to their restricted expression. To date, a number of protein candidates have emerged as potential clinical tools in the treatment of prostate cancer. Discovered over ten year ago, prostate stem cell antigen (PSCA) is a cell surface antigen that belongs to the Ly-6/Thy-1 family of glycosylphosphatidylinositol-anchored proteins. PSCA is highly overexpressed in human prostate cancer, with limited expression in normal tissues, making it an ideal target for both diagnosis and therapy. Several studies have now clearly correlated the expression of PSCA with relevant clinical benchmarks, such as Gleason score and metastasis, while others have demonstrated the efficacy of PSCA targeting in treatment through various modalities. The purpose of this review is to present the current body of knowledge about PSCA and its potential role in the treatment of human prostate cancer. PMID:18838214

Targeted delivery of antigen processing inhibitors to antigen presenting cells via mannose receptors.

PubMed

Raiber, Eun-Ang; Tulone, Calogero; Zhang, Yanjing; Martinez-Pomares, Luisa; Steed, Emily; Sponaas, Anna M; Langhorne, Jean; Noursadeghi, Mahdad; Chain, Benjamin M; Tabor, Alethea B

2010-05-21

Improved chemical inhibitors are required to dissect the role of specific antigen processing enzymes and to complement genetic models. In this study we explore the in vitro and in vivo properties of a novel class of targeted inhibitor of aspartic proteinases, in which pepstatin is coupled to mannosylated albumin (MPC6), creating an inhibitor with improved solubility and the potential for selective cell tropism. Using these compounds, we have demonstrated that MPC6 is taken up via mannose receptor facilitated endocytosis, leading to a slow but continuous accumulation of inhibitor within large endocytic vesicles within dendritic cells and a parallel inhibition of intracellular aspartic proteinase activity. Inhibition of intracellular proteinase activity is associated with reduction in antigen processing activity, but this is epitope-specific, preferentially inhibiting processing of T cell epitopes buried within compact proteinase-resistant protein domains. Unexpectedly, we have also demonstrated, using quenched fluorescent substrates, that little or no cleavage of the disulfide linker takes place within dendritic cells. This does not appear to affect the activity of MPC6 as an inhibitor of cathepsins D and E in vitro and in vivo. Finally, we have shown that MPC6 selectively targets dendritic cells and macrophages in spleen in vivo. Preliminary results suggest that access to nonlymphoid tissues is very limited in the steady state but is strongly enhanced at local sites of inflammation. The strategy adopted for MPC6 synthesis may therefore represent a more general way to deliver chemical inhibitors to cells of the innate immune system, especially at sites of inflammation.

Defensiveness, trait anxiety, and Epstein-Barr viral capsid antigen antibody titers in healthy college students.

PubMed

Esterling, B A; Antoni, M H; Kumar, M; Schneiderman, N

1993-03-01

The relationship of individual differences in repressive coping styles with differences in antibody titer to Epstein-Barr viral capsid antigen (EBV-VCA) were investigated in a normal, healthy college population made up of people previously exposed to EBV. Each of 54 1st-year undergraduates completed a battery of physical-status questions and items pertaining to potential behavioral immunomodulatory confounds, along with the Taylor Manifest Anxiety Scale (T-MAS) and the Marlowe-Crowne Social Desirability Scale (MC-SDS). Ss reporting high and middle levels of anxiety had higher antibody titers to EBV, suggesting poorer immune control over the latent virus, as compared with the low-anxious group. Similarly, high-defensive Ss had higher antibody titers than their low-defensive counterparts, and neither group differed from the middle group.

Cellular and humoral immune reactions in chronic active liver disease. II. Lymphocyte subsets and viral antigens in liver biopsies of patients with acute and chronic hepatitis B.

PubMed Central

Eggink, H F; Houthoff, H J; Huitema, S; Wolters, G; Poppema, S; Gips, C H

1984-01-01

The characteristics and distribution of the inflammatory infiltrate in liver biopsies of 25 patients with hepatitis B viral (HBV) infection were studied in relation to the distribution and expression of HBV antigens. Mononuclear subsets were characterized with monoclonal (OKT, OKM, Leu) antibodies to surface antigens. For the demonstration of viral antigens directly conjugated antibodies to surface (HBsAg), core (HBcAg) and 'e' (HBeAg) antigen were used. For the study of mutual relations all methods were performed on serial cut tissue sections. In chronic active hepatitis B (CAH-B, n = 12) OKT8+ lymphocytes of T cell origin were the only cell type present in areas with liver cell degeneration and T cell cytotoxicity appears to be the only immune mechanism. In chronic persistent hepatitis B (CPH-B, n = 7) the only conspicuous feature was the presence of many Leu 3+ lymphocytes of the helper/inducer population in the portal tracts. In acute hepatitis B (AHB, n = 6) OKT8+ cells of non-T origin (OKT1-,3-) and Leu 7+ cells of presumed natural killer (NK) potential predominated in the areas with liver cell necrosis, and non-T cell cytotoxicity appears to be the predominant immune mechanism. In none of these disease entities a positive spatial relation could be established between the cytotoxic cells and the demonstrable expression of HBV antigens in hepatocytes. It is concluded that differences in immunological reaction pattern may explain the different course in the three forms of HBV infection studied. Images Fig. 1 Fig. 2 PMID:6713726

Expression of potential target antigens for immunotherapy on primary and metastatic prostate cancers.

PubMed

Zhang, S; Zhang, H S; Reuter, V E; Slovin, S F; Scher, H I; Livingston, P O

1998-02-01

Defining the expression of tumor-associated antigens on primary and metastatic prostate cancer is the crucial first step in selecting appropriate targets for immune attack. In this study, the distribution of the tumor-associated antigens GM2, Tn, sTn, Thompson-Friedenreich antigen (TF), Globo H, Le(y), MUC1, MUC2, MUC3, MUC4, MUC5AC, MUC5B, MUC7, carcinoembryonic antigen, beta chain of human chorionic gonadotropin (hCG beta), HER2/neu, PSMA, and KSA on primary and metastatic prostate cancer and 16 types of normal tissues was compared by immunohistochemistry, using a panel of well-characterized monoclonal antibodies. Our results show that GM2, KSA, and MUC2 were strongly expressed on 8 or 9 of 9 metastatic prostate cancer biopsy specimens and, with PSMA, hCG beta, TF, Tn, and sTn, on 8 or more of 11 primary prostate cancer specimens. Tn, MUC1, and PSMA were expressed on 4-6 of 9 metastatic specimens. The remaining antigens were expressed on no more than three of nine metastatic specimens. Normal tissues were also tested with all antibodies. With regard to the eight antigens most widely expressed on prostate cancers, PSMA was not expressed significantly on any of the normal tissues except prostate epithelium. Tn, sTn, hCG beta, and MUC2 were detected on up to 3 of 10 types of normal epithelia. GM2, TF, MUC1, and KSA were more broadly distributed on normal epithelia, all primarily at the secretory borders. STn, KSA, and hCG beta were also detected in the testis, and GM2 was expressed on gray matter of brain. From the 30 antigens that we have screened, this study provides the basis for selecting GM2, TF, Tn, sTn, hCG beta, MUC1, MUC2, KSA, and PSMA as target antigens for specific immunotherapy of prostate cancer.

Diagnosis of viral hepatitis

PubMed Central

Easterbrook, Philippa J.; Roberts, Teri; Sands, Anita; Peeling, Rosanna

2017-01-01

Purpose of review Chronic hepatitis B virus (HBV) and hepatitis C virus (HCV) infections and HIV–HBV and HCV coinfection are major causes of chronic liver disease worldwide. Testing and diagnosis is the gateway for access to both treatment and prevention services, but there remains a large burden of undiagnosed infection globally. We review the global epidemiology, key challenges in the current hepatitis testing response, new tools to support the hepatitis global response (2016–2020 Global Hepatitis Health Sector strategy, and 2017 WHO guidelines on hepatitis testing) and future directions and innovations in hepatitis diagnostics. Recent findings Key challenges in the current hepatitis testing response include lack of quality-assured serological and low-cost virological in-vitro diagnostics, limited facilities for testing, inadequate data to guide country-specific hepatitis testing approaches, stigmatization of those with or at risk of viral hepatitis and lack of guidelines on hepatitis testing for resource-limited settings. The new Global Hepatitis Health Sector strategy sets out goals for elimination of viral hepatitis as a public health threat by 2030 and gives outcome targets for reductions in new infections and mortality, as well as service delivery targets that include testing, diagnosis and treatment. The 2017 WHO hepatitis testing guidelines for adults, adolescents and children in low-income and middle-income countries outline the public health approach to strengthen and expand current testing practices for viral hepatitis and addresses who to test (testing approaches), which serological and virological assays to use (testing strategies) as well as interventions to promote linkage to prevention and care. Summary Future directions and innovations in hepatitis testing include strategies to improve access such as through use of existing facility and community-based testing opportunities for hepatitis testing, near-patient or point-of-care assays for

Host and viral RNA-binding proteins involved in membrane targeting, replication and intercellular movement of plant RNA virus genomes

PubMed Central

Hyodo, Kiwamu; Kaido, Masanori; Okuno, Tetsuro

2014-01-01

Many plant viruses have positive-strand RNA [(+)RNA] as their genome. Therefore, it is not surprising that RNA-binding proteins (RBPs) play important roles during (+)RNA virus infection in host plants. Increasing evidence demonstrates that viral and host RBPs play critical roles in multiple steps of the viral life cycle, including translation and replication of viral genomic RNAs, and their intra- and intercellular movement. Although studies focusing on the RNA-binding activities of viral and host proteins, and their associations with membrane targeting, and intercellular movement of viral genomes have been limited to a few viruses, these studies have provided important insights into the molecular mechanisms underlying the replication and movement of viral genomic RNAs. In this review, we briefly overview the currently defined roles of viral and host RBPs whose RNA-binding activity have been confirmed experimentally in association with their membrane targeting, and intercellular movement of plant RNA virus genomes. PMID:25071804

Antigen-Specific CD8+ T Cells Fail To Respond to Shigella flexneri ▿

PubMed Central

Jehl, Stephanie P.; Doling, Amy M.; Giddings, Kara S.; Phalipon, Armelle; Sansonetti, Philippe J.; Goldberg, Marcia B.; Starnbach, Michael N.

2011-01-01

CD8+ T lymphocytes often play a primary role in adaptive immunity to cytosolic microbial pathogens. Surprisingly, CD8+ T cells are not required for protective immunity to the enteric pathogen Shigella flexneri, despite the ability of Shigella to actively secrete proteins into the host cytoplasm, a location from which antigenic peptides are processed for presentation to CD8+ T cells. To determine why CD8+ T cells fail to play a role in adaptive immunity to S. flexneri, we investigated whether antigen-specific CD8+ T cells are primed during infection but are unable to confer protection or, alternatively, whether T cells fail to be primed. To test whether Shigella is capable of stimulating an antigen-specific CD8+ T-cell response, we created an S. flexneri strain that constitutively secretes a viral CD8+ T-cell epitope via the Shigella type III secretion system and characterized the CD8+ T-cell response to this strain both in mice and in cultured cells. Surprisingly, no T cells specific for the viral epitope were stimulated in mice infected with this strain, and cells infected with the recombinant strain were not targeted by epitope-specific T cells. Additionally, we found that the usually robust T-cell response to antigens artificially introduced into the cytoplasm of cultured cells was significantly reduced when the antigen-presenting cell was infected with Shigella. Collectively, these results suggest that antigen-specific CD8+ T cells are not primed during S. flexneri infection and, as a result, afford little protection to the host during primary or subsequent infection. PMID:21357720

Targeting Antigens to Dec-205 on Dendritic Cells Induces Immune Protection in Experimental Colitis in Mice

PubMed Central

Wadwa, Munisch; Klopfleisch, Robert; Buer, Jan; Westendorf, Astrid M.

2016-01-01

The endocytotic c-type lectin receptor DEC-205 is highly expressed on immature dendritic cells. In previous studies, it was shown that antigen-targeting to DEC-205 is a useful tool for the induction of antigen-specific Foxp3+ regulatory T cells and thereby can prevent inflammatory processes. However, whether this approach is sufficient to mediate tolerance in mucosal tissues like the gut is unknown. In this study, we established a new mouse model in which the adoptive transfer of naive hemagglutinin (HA)-specific CD4+Foxp3– T cells into VILLIN-HA transgenic mice leads to severe colitis. To analyze if antigen-targeting to DEC-205 could protect against inflammation of the gut, VILLIN-HA transgenic mice were injected with an antibody–antigen complex consisting of the immunogenic HA110–120 peptide coupled to an α-DEC-205 antibody (DEC-HA) before adoptive T cell transfer. DEC-HA-treated mice showed significantly less signs of intestinal inflammation as was demonstrated by reduced loss of body weight and histopathology in the gut. Strikingly, abrogated intestinal inflammation was mediated via the conversion of naive HA-specific CD4+Foxp3– T cells into HA-specific CD4+Foxp3+ regulatory T cells. In this study, we provide evidence that antigen-targeting to DEC-205 can be utilized for the induction of tolerance in mucosal organs that are confronted with large numbers of exogenous antigens. PMID:27141310

Mathematical models of immune effector responses to viral infections: Virus control versus the development of pathology

NASA Astrophysics Data System (ADS)

Wodarz, Dominik

2005-12-01

This article reviews mathematical models which have investigated the importance of lytic and non-lytic immune responses for the control of viral infections. Lytic immune responses fight the virus by killing infected cells, while non-lytic immune responses fight the virus by inhibiting viral replication while leaving the infected cell alive. The models suggest which types or combinations of immune responses are required to resolve infections which vary in their characteristics, such as the rate of viral replication and the rate of virus-induced target cell death. This framework is then applied to persistent infections and viral evolution. It is investigated how viral evolution and antigenic escape can influence the relative balance of lytic and non-lytic responses over time, and how this might correlate with the transition from an asymptomatic infection to pathology. This is discussed in the specific context of hepatitis C virus infection.

Simultaneous targeting of prostate stem cell antigen and prostate-specific membrane antigen improves the killing of prostate cancer cells using a novel modular T cell-retargeting system.

PubMed

Arndt, Claudia; Feldmann, Anja; Koristka, Stefanie; Cartellieri, Marc; Dimmel, Maria; Ehninger, Armin; Ehninger, Gerhard; Bachmann, Michael

2014-09-01

Recently, we described a novel modular platform technology in which T cell-recruitment and tumor-targeting domains of conventional bispecific antibodies are split to independent components, a universal effector module (EM) and replaceable monospecific/monovalent target modules (TMs) that form highly efficient T cell-retargeting complexes. Theoretically, our unique strategy should allow us to simultaneously retarget T cells to different tumor antigens by combining the EM with two or more different monovalent/monospecific TMs or even with bivalent/bispecific TMs, thereby overcoming limitations of a monospecific treatment such as the selection of target-negative tumor escape variants. In order to advance our recently introduced prostate stem cell antigen (PSCA)-specific modular system for a dual-targeting of prostate cancer cells, two additional TMs were constructed: a monovalent/monospecific TM directed against the prostate-specific membrane antigen (PSMA) and a bivalent/bispecific TM (bsTM) with specificity for PSMA and PSCA. The functionality of the novel dual-targeting strategies was analyzed by performing T cell activation and chromium release assays. Similar to the PSCA-specific modular system, the novel PSMA-specific modular system mediates an efficient target-dependent and -specific tumor cell lysis at low E:T ratios and picomolar Ab concentrations. Moreover, by combination of the EM with either the bispecific TM directed to PSMA and PSCA or both monospecifc TMs directed to either PSCA or PSMA, dual-specific targeting complexes were formed which allowed us to kill potential escape variants expressing only one or the other target antigen. Overall, the novel modular system represents a promising tool for multiple tumor targeting. © 2014 Wiley Periodicals, Inc.

Antigen sensitivity of CD22-specific chimeric T cell receptors is modulated by target epitope distance from the cell membrane

PubMed Central

James, Scott E.; Greenberg, Philip D.; Jensen, Michael C.; Lin, Yukang; Wang, Jinjuan; Till, Brian G.; Raubitschek, Andrew A.; Forman, Stephen J.; Press, Oliver W.

2008-01-01

We have targeted CD22 as a novel tumor-associated antigen for recognition by human CTL genetically modified to express chimeric T cell receptors (cTCR) recognizing this surface molecule. CD22-specifc cTCR targeting different epitopes of the CD22 molecule promoted efficient lysis of target cells expressing high levels of CD22 with a maximum lytic potential that appeared to decrease as the distance of the target epitope from the target cell membrane increased. Targeting membrane-distal CD22 epitopes with cTCR+ CTL revealed defects in both degranulation and lytic granule targeting. CD22-specific cTCR+ CTL exhibited lower levels of maximum lysis and lower antigen sensitivity than CTL targeting CD20, which has a shorter extracellular domain than CD22. This diminished sensitivity was not a result of reduced avidity of antigen engagement, but instead reflected weaker signaling per triggered cTCR molecule when targeting membrane-distal epitopes of CD22. Both of these parameters were restored by targeting a ligand expressing the same epitope but constructed as a truncated CD22 molecule to approximate the length of a TCR:pMHC complex. The reduced sensitivity of CD22-specific cTCR+ CTL for antigen-induced triggering of effector functions has potential therapeutic applications, as such cells selectively lysed B cell lymphoma lines expressing high levels of CD22 but demonstrated minimal activity against autologous normal B cells, which express lower levels of CD22. Thus, our results demonstrate that cTCR signal strength – and consequently antigen sensitivity – can be modulated by differential choice of target epitopes with respect to distance from the cell membrane, allowing discrimination between targets with disparate antigen density. PMID:18453625

Strategies to induce broadly protective antibody responses to viral glycoproteins.

PubMed

Krammer, F

2017-05-01

Currently, several universal/broadly protective influenza virus vaccine candidates are under development. Many of these vaccines are based on strategies to induce protective antibody responses against the surface glycoproteins of antigenically and genetically diverse influenza viruses. These strategies might also be applicable to surface glycoproteins of a broad range of other important viral pathogens. Areas covered: Common strategies include sequential vaccination with divergent antigens, multivalent approaches, vaccination with glycan-modified antigens, vaccination with minimal antigens and vaccination with antigens that have centralized/optimized sequences. Here we review these strategies and the underlying concepts. Furthermore, challenges, feasibility and applicability to other viral pathogens are discussed. Expert commentary: Several broadly protective/universal influenza virus vaccine strategies will be tested in humans in the coming years. If successful in terms of safety and immunological readouts, they will move forward into efficacy trials. In the meantime, successful vaccine strategies might also be applied to other antigenically diverse viruses of concern.

Inhibition of adenovirus multiplication by short interfering RNAs directly or indirectly targeting the viral DNA replication machinery.

PubMed

Kneidinger, Doris; Ibrišimović, Mirza; Lion, Thomas; Klein, Reinhard

2012-06-01

Human adenoviruses are a common threat to immunocompromised patients, e.g., HIV-positive individuals or solid-organ and, in particular, allogeneic stem cell transplant recipients. Antiviral drugs have a limited effect on adenoviruses, and existing treatment modalities often fail to prevent fatal outcome. Silencing of viral genes by short interfering RNAs (siRNAs) holds a great promise in the treatment of viral infections. The aim of the present study was to identify adenoviral candidate targets for RNA interference-mediated inhibition of adenoviral replication. We investigated the impact of silencing of a set of early, middle, and late viral genes on the replication of adenovirus 5 in vitro. Adenovirus replication was inhibited by siRNAs directed against the adenoviral E1A, DNA polymerase, preterminal protein (pTP), IVa2, hexon, and protease genes. Silencing of early and middle genes was more effective in inhibiting adenovirus multiplication than was silencing of late genes. A siRNA directed against the viral DNA polymerase mRNA decreased viral genome copy numbers and infectious virus progeny by several orders of magnitude. Since silencing of any of the early genes directly or indirectly affected viral DNA synthesis, our data suggest that reducing viral genome copy numbers is a more promising strategy for the treatment of adenoviral infections than is reducing the numbers of proteins necessary for capsid generation. Thus, adenoviral DNA replication was identified as a key target for RNAi-mediated inhibition of adenovirus multiplication. In addition, the E1A transcripts emerged as a second important target, because its knockdown markedly improved the viability of cells at late stages of infection. Copyright © 2012 Elsevier B.V. All rights reserved.

Targeting of tumor-associated antigens (TAA) in experimental immunotherapy

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ravikumar, T.S.; Galbo, L.; Marini, C.

1986-06-01

We have previously shown the superiority of tumor-associated antigens (TAA) to function as effective immunogens when administered with bilayer membrane vesicles called liposomes. The ability of liposomes to target TAA to host antigen-presenting cells is analyzed here. 1-Butanol extracted TAA from two syngeneic rat colon cancer tumors (WB 2054 and W 1756) was radioiodinated (/sup 131/I-TAA). Free /sup 131/I and /sup 131/I-TAA (2.8 X 10(7) cpm and 75 micrograms TAA per rat) were used as tracers, with or without incorporation into liposomes (composition: sphingomyelin, cholesterol, dicetyl phosphate at 70:24:6 molar ratio). Six groups of male rats (BN X WF formore » WB2054 and Wistar/Furth for W1756, n = 18 each group) were injected iv with either free tracers or the tracers incorporated into liposomes. Whole blood clearance curve was biphasic (half-life alpha = 5 min; half life beta = 12 hr), suggesting a two-compartmental model of distribution. Seven animals from each group were sacrificed at set times (15 min to 48 hr), organs harvested and cpm/g of tissue estimated. Liposome /sup 131/I and liposome /sup 131/I-TAA were targeted to and retained preferentially in liver and spleen. Four animals from each group were imaged serially using a gamma camera. Matched pair analysis of regions showed persistently higher activity in liver-spleen area when liposomes were used (P less than 0.001). The uptake of radiolabeled antigens by plastic adherent mononuclear cells in liver and spleen was significantly higher when presented with liposomes (macrophage uptake index: liver = 1.65 vs 0.55; spleen = 5.85 vs 1.15; with and without liposomes, respectively).« less

Adenovirus-Mediated Gene Therapy Against Viral Biothreat Agents

DTIC Science & Technology

2016-04-12

economy. Vaccine development is an important strategy to thwart the threat of these viral biothreat agents. There is an urgent need to improve...Alberta, Tl A 8K6. Canada E-mail: josh. wu@drdc-rddc.gc.ca .• 78 JoshQ.H. Wu existing vaccines against these agents and to develop new ones. Gene...of vaccines against viral biothreat agents. Genes encoding protective antigens of viral biothreat agents can be carried by these viral vectors and

M cell-targeting strategy facilitates mucosal immune response and enhances protection against CVB3-induced viral myocarditis elicited by chitosan-DNA vaccine.

PubMed

Ye, Ting; Yue, Yan; Fan, Xiangmei; Dong, Chunsheng; Xu, Wei; Xiong, Sidong

2014-07-31

Efficient delivery of antigen to mucosal associated lymphoid tissue is a first and critical step for successful induction of mucosal immunity by vaccines. Considering its potential transcytotic capability, M cell has become a more and more attractive target for mucosal vaccines. In this research, we designed an M cell-targeting strategy by which mucosal delivery system chitosan (CS) was endowed with M cell-targeting ability via conjugating with a CPE30 peptide, C terminal 30 amino acids of clostridium perfringens enterotoxin (CPE), and then evaluated its immune-enhancing ability in the context of coxsackievirus B3 (CVB3)-specific mucosal vaccine consisting of CS and a plasmid encoding CVB3 predominant antigen VP1. It had shown that similar to CS-pVP1, M cell-targeting CPE30-CS-pVP1 vaccine appeared a uniform spherical shape with about 300 nm diameter and +22 mV zeta potential, and could efficiently protect DNA from DNase I digestion. Mice were orally immunized with 4 doses of CPE30-CS-pVP1 containing 50 μg pVP1 at 2-week intervals and challenged with CVB3 4 weeks after the last immunization. Compared with CS-pVP1 vaccine, CPE30-CS-pVP1 vaccine had no obvious impact on CVB3-specific serum IgG level and splenic T cell immune responses, but significantly increased specific fecal SIgA level and augmented mucosal T cell immune responses. Consequently, much milder myocarditis and lower viral load were witnessed in CPE30-CS-pVP1 immunized group. The enhanced immunogenicity and immunoprotection were associated with the M cell-targeting ability of CPE30-CS-pVP1 which improved its mucosal uptake and transcytosis. Our findings indicated that CPE30-CS-pVP1 may represent a novel prophylactic vaccine against CVB3-induced myocarditis, and this M cell-targeting strategy indeed could be applied as a promising and universal platform for mucosal vaccine development. Copyright © 2014 Elsevier Ltd. All rights reserved.

Association of human leukocyte antigen class II allele and haplotypes in chikungunya viral infection in a western Indian population.

PubMed

Thanapati, Subrat; Hande, Aparna; Das, Rumki; Gurav, Yogesh; Tripathy, Anuradha S

2014-05-01

Genes coding for human leukocyte antigen (HLA) class II molecules are polymorphic and have been shown to influence susceptibility to viral diseases. One hundred patients with acute chikungunya with and without viral load and 250 chikungunya negative controls from western India were studied for the distribution of HLA class II alleles by PCR with sequence-specific primer (SSP) method. Frequency of DRB1*11 allele group (patients vs controls: p=0.002, Pc=0.036, OR=0.21) and haplotype DRB1*11/DQB1*03 (patients vs controls: p=0.007, OR=0.15) were significantly low, while haplotype DRB1*04/DQB1*03 (patients vs controls: p=0.042, OR=1.94) was significantly high in the patient population. HLA DQB1*04 allele was found only in the patient group with viral load (n=17), suggesting possible involvement of the same with chikungunya virus (CHIKV) replication. Association of HLA-DRB1*11 and the emergence of DRB1*11/DQB1*03 & DRB1*04/DQB1*03 as resistant and susceptible haplotypes towards CHIKV infection is being reported for the first time. Our results suggest that genetic susceptibility and/or resistance to chikungunya infection may be modulated by HLA class II alleles.

Towards peptide vaccines against Zika virus: Immunoinformatics combined with molecular dynamics simulations to predict antigenic epitopes of Zika viral proteins.

PubMed

Usman Mirza, Muhammad; Rafique, Shazia; Ali, Amjad; Munir, Mobeen; Ikram, Nazia; Manan, Abdul; Salo-Ahen, Outi M H; Idrees, Muhammad

2016-12-09

The recent outbreak of Zika virus (ZIKV) infection in Brazil has developed to a global health concern due to its likely association with birth defects (primary microcephaly) and neurological complications. Consequently, there is an urgent need to develop a vaccine to prevent or a medicine to treat the infection. In this study, immunoinformatics approach was employed to predict antigenic epitopes of Zika viral proteins to aid in development of a peptide vaccine against ZIKV. Both linear and conformational B-cell epitopes as well as cytotoxic T-lymphocyte (CTL) epitopes were predicted for ZIKV Envelope (E), NS3 and NS5 proteins. We further investigated the binding interactions of altogether 15 antigenic CTL epitopes with three class I major histocompatibility complex (MHC I) proteins after docking the peptides to the binding groove of the MHC I proteins. The stability of the resulting peptide-MHC I complexes was further studied by molecular dynamics simulations. The simulation results highlight the limits of rigid-body docking methods. Some of the antigenic epitopes predicted and analyzed in this work might present a preliminary set of peptides for future vaccine development against ZIKV.

Adoptive immunotherapy for B-cell malignancies with autologous chimeric antigen receptor modified tumor targeted T cells.

PubMed

Park, Jae H; Brentjens, Renier J

2010-04-01

Chemotherapy-resistant B-cell hematologic malignancies may be cured with allogeneic hematopoietic stem cell transplantation (HSCT), demonstrating the potential susceptibility of these tumors to donor T-cell mediated immune responses. However, high rates of transplant-related morbidity and mortality limit this approach. For this reason, there is an urgent need for less-toxic forms of immune-based cellular therapy to treat these malignancies. Adoptive transfer of autologous T cells genetically modified to express chimeric antigen receptors (CARs) targeted to specific tumor-associated antigens represents an attractive means of overcoming the limitations of conventional HSCT. To this end, investigators have generated CARs targeted to various antigens expressed by B-cell malignancies, optimized the design of these CARs to enhance receptor mediated T cell signaling, and demonstrated significant anti-tumor efficacy of the resulting CAR modified T cells both in vitro and in vivo mouse tumor models. These encouraging preclinical data have justified the translation of this approach to the clinical setting with currently 12 open clinical trials and one completed clinical trial treating various B-cell malignancies utilizing CAR modified T cells targeted to either the CD19 or CD20 B-cell specific antigens.

Eicosanoids and Respiratory Viral Infection: Coordinators of Inflammation and Potential Therapeutic Targets

PubMed Central

McCarthy, Mary K.; Weinberg, Jason B.

2012-01-01

Viruses are frequent causes of respiratory infection, and viral respiratory infections are significant causes of hospitalization, morbidity, and sometimes mortality in a variety of patient populations. Lung inflammation induced by infection with common respiratory pathogens such as influenza and respiratory syncytial virus is accompanied by increased lung production of prostaglandins and leukotrienes, lipid mediators with a wide range of effects on host immune function. Deficiency or pharmacologic inhibition of prostaglandin and leukotriene production often results in a dampened inflammatory response to acute infection with a respiratory virus. These mediators may, therefore, serve as appealing therapeutic targets for disease caused by respiratory viral infection. PMID:22665949

Antigen processing and remodeling of the endosomal pathway: requirements for antigen cross-presentation.

PubMed

Compeer, Ewoud Bernardus; Flinsenberg, Thijs Willem Hendrik; van der Grein, Susanna Geertje; Boes, Marianne

2012-01-01

Cross-presentation of endocytosed antigen as peptide/class I major histocompatibility complex complexes plays a central role in the elicitation of CD8(+) T cell clones that mediate anti-viral and anti-tumor immune responses. While it has been clear that there are specific subsets of professional antigen presenting cells capable of antigen cross-presentation, identification of mechanisms involved is still ongoing. Especially amongst dendritic cells (DC), there are specialized subsets that are highly proficient at antigen cross-presentation. We here present a focused survey on the cell biological processes in the endosomal pathway that support antigen cross-presentation. This review highlights DC-intrinsic mechanisms that facilitate the cross-presentation of endocytosed antigen, including receptor-mediated uptake, maturation-induced endosomal sorting of membrane proteins, dynamic remodeling of endosomal structures and cell surface-directed endosomal trafficking. We will conclude with the description of pathogen-induced deviation of endosomal processing, and discuss how immune evasion strategies pertaining endosomal trafficking may preclude antigen cross-presentation.

Engineering tolerance using biomaterials to target and control antigen presenting cells.

PubMed

Tostanoski, Lisa H; Gosselin, Emily A; Jewell, Christopher M

2016-05-01

Autoimmune diseases occur when cells of the adaptive immune system incorrectly recognize and attack "self" tissues. Importantly, the proliferation and differentiation of these cells is triggered and controlled by interactions with antigen presenting cells (APCs), such as dendritic cells. Thus, modulating the signals transduced by APCs (e.g., cytokines, costimulatory surface proteins) has emerged as a promising strategy to promote tolerance for diseases such as multiple sclerosis, type 1 diabetes, and lupus. However, many approaches have been hindered by non-specific activity of immunosuppressive or immunoregulatory cues, following systemic administration of soluble factors via traditional injections routes (e.g., subcutaneous, intravenous). Biomaterials offer a unique opportunity to control the delivery of tolerogenic signals in vivo via properties such as controlled particle size, tunable release kinetics, and co-delivery of multiple classes of cargo. In this review, we highlight recent reports that exploit these properties of biomaterials to target APCs and promote tolerance via three strategies, i) passive or active targeting of particulate carriers to APCs, ii) biomaterial-mediated control over antigen localization and processing, and iii) targeted delivery of encapsulated or adsorbed immunomodulatory signals. These reports represent exciting advances toward the goal of more effective therapies for autoimmune diseases, without the broad suppressive effects associated with current clinically-approved therapies.

Antigenic variation: Molecular and genetic mechanisms of relapsing disease

DOE Office of Scientific and Technical Information (OSTI.GOV)

Cruse, J.M.; Lewis, R.E.

1987-01-01

This book contains 10 chapters. They are: Contemporary Concepts of Antigenic Variation; Antigenic Variation in the Influenza Viruses; Mechanisms of Escape of Visna Lentiviruses from Immunological Control; A Review of Antigenic Variation by the Equine Infectious Anemia Virus; Biologic and Molecular Variations in AIDS Retrovirus Isolates; Rabies Virus Infection: Genetic Mutations and the Impact on Viral Pathogenicity and Immunity; Immunobiology of Relapsing Fever; Antigenic Variation in African Trypanosomes; Antigenic Variation and Antigenic Diversity in Malaria; and Mechanisms of Immune Evasion in Schistosomiasis.

A high molecular weight-melanoma associated antigen-specific chimeric antigen receptor redirects lymphocytes to target human melanomas

PubMed Central

Burns, William R.; Zhao, Yangbing; Frankel, Timothy L.; Hinrichs, Christian S.; Zheng, Zhili; Xu, Hui; Feldman, Steven A.; Ferrone, Soldano; Rosenberg, Steven A.; Morgan, Richard A.

2011-01-01

Immunotherapy, particularly the adoptive cell transfer (ACT) of tumor infiltrating lymphocytes (TIL), is a very promising therapy for metastatic melanoma. Some patients unable to receive TIL have been successfully treated with autologous peripheral blood lymphocytes (PBL), genetically modified to express HLA class I antigen restricted, melanoma antigen-reactive T-cell receptors; however, substantial numbers of patients remain ineligible due to the lack of expression of the restricting HLA class I allele. We sought to overcome this limitation by designing a non-MHC-restricted, chimeric antigen receptor (CAR) targeting the high molecular weight-melanoma associated antigen (HMW-MAA), which is highly expressed on over 90% of human melanomas but has a restricted distribution in normal tissues. HMW-MAA-specific CARs containing an antigen recognition domain based on variations of the HMW-MAA-specific monoclonal antibody (mAb) 225.28S and a T-cell activation domain based on combinations of CD28, 4-1BB, and CD3ζ activation motifs were constructed within a retroviral vector to allow stable gene transfer into cells and their progeny. Following optimization of the HMW-MAA-specific CAR for expression and function in human PBL, these gene-modified T cells secreted cytokines, were cytolytic, and proliferated in response to HMW-MAA expressing cell lines. Furthermore, the receptor functioned in both CD4+ and CD8+ cells, was non-MHC-restricted, and reacted against explanted human melanomas. To evaluate this HMW-MAA-specific CAR in patients with metastatic melanoma, we developed a clinical-grade retroviral packaging line. This may represent a novel means to treat the majority of patients with advanced melanoma, most notably those unable to receive current ACT therapies. PMID:20395199

Structural flexibility at a major conserved antibody target on hepatitis C virus E2 antigen.

PubMed

Kong, Leopold; Lee, David E; Kadam, Rameshwar U; Liu, Tong; Giang, Erick; Nieusma, Travis; Garces, Fernando; Tzarum, Netanel; Woods, Virgil L; Ward, Andrew B; Li, Sheng; Wilson, Ian A; Law, Mansun

2016-10-24

Hepatitis C virus (HCV) is a major cause of liver disease, affecting over 2% of the world's population. The HCV envelope glycoproteins E1 and E2 mediate viral entry, with E2 being the main target of neutralizing antibody responses. Structural investigations of E2 have produced templates for vaccine design, including the conserved CD81 receptor-binding site (CD81bs) that is a key target of broadly neutralizing antibodies (bNAbs). Unfortunately, immunization with recombinant E2 and E1E2 rarely elicits sufficient levels of bNAbs for protection. To understand the challenges for eliciting bNAb responses against the CD81bs, we investigated the E2 CD81bs by electron microscopy (EM), hydrogen-deuterium exchange (HDX), molecular dynamics (MD), and calorimetry. By EM, we observed that HCV1, a bNAb recognizing the N-terminal region of the CD81bs, bound a soluble E2 core construct from multiple angles of approach, suggesting components of the CD81bs are flexible. HDX of multiple E2 constructs consistently indicated the entire CD81bs was flexible relative to the rest of the E2 protein, which was further confirmed by MD simulations. However, E2 has a high melting temperature of 84.8 °C, which is more akin to proteins from thermophilic organisms. Thus, recombinant E2 is a highly stable protein overall, but with an exceptionally flexible CD81bs. Such flexibility may promote induction of nonneutralizing antibodies over bNAbs to E2 CD81bs, underscoring the necessity of rigidifying this antigenic region as a target for rational vaccine design.

T cells targeting a neuronal paraneoplastic antigen mediate tumor rejection and trigger CNS autoimmunity with humoral activation.

PubMed

Blachère, Nathalie E; Orange, Dana E; Santomasso, Bianca D; Doerner, Jessica; Foo, Patricia K; Herre, Margaret; Fak, John; Monette, Sébastien; Gantman, Emily C; Frank, Mayu O; Darnell, Robert B

2014-11-01

Paraneoplastic neurologic diseases (PND) involving immune responses directed toward intracellular antigens are poorly understood. Here, we examine immunity to the PND antigen Nova2, which is expressed exclusively in central nervous system (CNS) neurons. We hypothesized that ectopic expression of neuronal antigen in the periphery could incite PND. In our C57BL/6 mouse model, CNS antigen expression limits antigen-specific CD4+ and CD8+ T-cell expansion. Chimera experiments demonstrate that this tolerance is mediated by antigen expression in nonhematopoietic cells. CNS antigen expression does not limit tumor rejection by adoptively transferred transgenic T cells but does limit the generation of a memory population that can be expanded upon secondary challenge in vivo. Despite mediating cancer rejection, adoptively transferred transgenic T cells do not lead to paraneoplastic neuronal targeting. Preliminary experiments suggest an additional requirement for humoral activation to induce CNS autoimmunity. This work provides evidence that the requirements for cancer immunity and neuronal autoimmunity are uncoupled. Since humoral immunity was not required for tumor rejection, B-cell targeting therapy, such as rituximab, may be a rational treatment option for PND that does not hamper tumor immunity. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Merkel cell carcinoma: histopathologic and prognostic features according to the immunohistochemical expression of Merkel cell polyomavirus large T antigen correlated with viral load.

PubMed

Leroux-Kozal, Valérie; Lévêque, Nicolas; Brodard, Véronique; Lesage, Candice; Dudez, Oriane; Makeieff, Marc; Kanagaratnam, Lukshe; Diebold, Marie-Danièle

2015-03-01

Merkel cell carcinoma (MCC) is a neuroendocrine skin malignancy frequently associated with Merkel cell polyomavirus (MCPyV), which is suspected to be oncogenic. In a series of MCC patients, we compared clinical, histopathologic, and prognostic features according to the expression of viral large T antigen (LTA) correlated with viral load. We evaluated the LTA expression by immunohistochemistry using CM2B4 antibody and quantified viral load by real-time polymerase chain reaction. We analyzed formalin-fixed, paraffin-embedded (FFPE) tissue samples (n = 36) and corresponding fresh-frozen biopsies when available (n = 12), of the primary tumor and/or metastasis from 24 patients. MCPyV was detected in 88% and 58% of MCC patients by real-time polymerase chain reaction and immunohistochemistry, respectively. The relevance of viral load measurements was demonstrated by the strong consistency of viral load level between FFPE and corresponding frozen tissues as well as between primary tumor and metastases. From FFPE samples, 2 MCC subgroups were distinguished based on a viral load threshold defined by the positivity of CM2B4 immunostaining. In the LTA-negative subgroup with no or low viral load (nonsignificant), tumor cells showed more anisokaryosis (P = .01), and a solar elastosis around the tumor was more frequently observed (P = .03). LTA-positive MCCs with significant viral load had a lower proliferation index (P = .03) and a longer survival of corresponding patients (P = .008). Depending on MCPyV involvement, 2 MCC subgroups can be distinguished on histopathologic criteria, and the CM2B4 antibody is able to differentiate them reliably. Furthermore, the presence of a significant viral load in tumors is predictive of better prognosis. Copyright © 2015 Elsevier Inc. All rights reserved.

Plum Pox Virus 6K1 Protein Is Required for Viral Replication and Targets the Viral Replication Complex at the Early Stage of Infection.

PubMed

Cui, Hongguang; Wang, Aiming

2016-05-15

The potyviral RNA genome encodes two polyproteins that are proteolytically processed by three viral protease domains into 11 mature proteins. Extensive molecular studies have identified functions for the majority of the viral proteins. For example, 6K2, one of the two smallest potyviral proteins, is an integral membrane protein and induces the endoplasmic reticulum (ER)-originated replication vesicles that target the chloroplast for robust viral replication. However, the functional role of 6K1, the other smallest protein, remains uncharacterized. In this study, we developed a series of recombinant full-length viral cDNA clones derived from a Canadian Plum pox virus (PPV) isolate. We found that deletion of any of the short motifs of 6K1 (each of which ranged from 5 to 13 amino acids), most of the 6K1 sequence (but with the conserved sequence of the cleavage sites being retained), or all of the 6K1 sequence in the PPV infectious clone abolished viral replication. The trans expression of 6K1 or the cis expression of a dislocated 6K1 failed to rescue the loss-of-replication phenotype, suggesting the temporal and spatial requirement of 6K1 for viral replication. Disruption of the N- or C-terminal cleavage site of 6K1, which prevented the release of 6K1 from the polyprotein, either partially or completely inhibited viral replication, suggesting the functional importance of the mature 6K1. We further found that green fluorescent protein-tagged 6K1 formed punctate inclusions at the viral early infection stage and colocalized with chloroplast-bound viral replicase elements 6K2 and NIb. Taken together, our results suggest that 6K1 is required for viral replication and is an important viral element of the viral replication complex at the early infection stage. Potyviruses account for more than 30% of known plant viruses and consist of many agriculturally important viruses. The genomes of potyviruses encode two polyproteins that are proteolytically processed into 11 mature

Plum Pox Virus 6K1 Protein Is Required for Viral Replication and Targets the Viral Replication Complex at the Early Stage of Infection

PubMed Central

Cui, Hongguang

2016-01-01

ABSTRACT The potyviral RNA genome encodes two polyproteins that are proteolytically processed by three viral protease domains into 11 mature proteins. Extensive molecular studies have identified functions for the majority of the viral proteins. For example, 6K2, one of the two smallest potyviral proteins, is an integral membrane protein and induces the endoplasmic reticulum (ER)-originated replication vesicles that target the chloroplast for robust viral replication. However, the functional role of 6K1, the other smallest protein, remains uncharacterized. In this study, we developed a series of recombinant full-length viral cDNA clones derived from a Canadian Plum pox virus (PPV) isolate. We found that deletion of any of the short motifs of 6K1 (each of which ranged from 5 to 13 amino acids), most of the 6K1 sequence (but with the conserved sequence of the cleavage sites being retained), or all of the 6K1 sequence in the PPV infectious clone abolished viral replication. The trans expression of 6K1 or the cis expression of a dislocated 6K1 failed to rescue the loss-of-replication phenotype, suggesting the temporal and spatial requirement of 6K1 for viral replication. Disruption of the N- or C-terminal cleavage site of 6K1, which prevented the release of 6K1 from the polyprotein, either partially or completely inhibited viral replication, suggesting the functional importance of the mature 6K1. We further found that green fluorescent protein-tagged 6K1 formed punctate inclusions at the viral early infection stage and colocalized with chloroplast-bound viral replicase elements 6K2 and NIb. Taken together, our results suggest that 6K1 is required for viral replication and is an important viral element of the viral replication complex at the early infection stage. IMPORTANCE Potyviruses account for more than 30% of known plant viruses and consist of many agriculturally important viruses. The genomes of potyviruses encode two polyproteins that are proteolytically

African swine fever virus serotype-specific proteins are significant protective antigens for African swine fever.

PubMed

Burmakina, G; Malogolovkin, A; Tulman, E R; Zsak, L; Delhon, G; Diel, D G; Shobogorov, N M; Morgunov, Yu P; Morgunov, S Yu; Kutish, G F; Kolbasov, D; Rock, D L

2016-07-01

African swine fever (ASF) is an emerging disease threat for the swine industry worldwide. No ASF vaccine is available and progress is hindered by lack of knowledge concerning the extent of ASFV strain diversity and the viral antigens conferring type-specific protective immunity in pigs. Available data from vaccination/challenge experiments in pigs indicate that ASF protective immunity may be haemadsorption inhibition (HAI) serotype-specific. Recently, we have shown that two ASFV proteins, CD2v (EP402R) and C-type lectin (EP153R), are necessary and sufficient for mediating HAI serological specificity (Malogolovkin et al., 2015).. Here, using ASFV inter-serotypic chimeric viruses and vaccination/challenge experiments in pigs, we demonstrate that serotype-specific CD2v and/or C-type lectin proteins are important for protection against homologous ASFV infection. Thus, these viral proteins represent significant protective antigens for ASFV that should be targeted in future vaccine design and development. Additionally, these data support the concept of HAI serotype-specific protective immunity.

Viral-induced Modulation of Multiple Checkpoint Proteins in Cancers.

PubMed

Nuovo, Gerard J; Folcik, Virginia A; Magro, Cynthia

2017-07-01

Therapy with checkpoint inhibitors represents a major advance in cancer treatment. The purpose of this study was to examine the expression patterns of the checkpoint proteins programmed death ligand 1 (PD L1), PD L2, indoleamine 2,3-dioxygenase 1 (IDO1), and cytotoxic T-lymphocyte antigen 4 (CTLA4) in cancers including those associated with viral infections. Normal, noninflamed tissues rarely express checkpoint proteins with exceptions including the placenta and stomach. Expression of PD L1 was noted in 30%, PD L2 in 18%, IDO1 in 13%, and CTLA4 in 14% of 333 nonviral malignancies including endometrial, ovarian, lung, and breast cancers. The expression of each checkpoint protein was significantly higher among 166 cases of viral-related (mostly human papillomavirus) cancers where expression of PD L1 was noted in 84%, PD L2 in 67%, IDO1 in 61%, and CTLA4 in 37% (each P value <0.001); 97% of the viral-related cancers showed expression of at least 1 checkpoint protein. In addition, over 90% of the CD8 cells in the viral-associated cancers were quiescent based on low coexpression of Ki-67 as well as pSTAT1. It is concluded that viral infection in cancers is associated with the increased expression of key checkpoint proteins. This indicates that cancers with productive viral infection may be better targets for checkpoint inhibitor therapy.

Simultaneous human platelet antigen genotyping and detection of novel single nucleotide polymorphisms by targeted next-generation sequencing.

PubMed

Davey, Sue; Navarrete, Cristina; Brown, Colin

2017-06-01

Twenty-nine human platelet antigen systems have been described to date, but the majority of current genotyping methods are restricted to the identification of those most commonly associated with alloantibody production in a clinical context. This can result in a protracted investigation if causative human platelet antigens are rare or novel. A targeted next-generation sequencing approach was designed to detect all known human platelet antigens with the additional capability of identifying novel mutations in the encoding genes. A targeted enrichment, high-sensitivity HaloPlex assay was designed to sequence all exons and flanking regions of the six genes known to encode human platelet antigens. Indexed DNA libraries were prepared from 47 previously human platelet antigen-genotyped samples and subsequently combined into one of three pools for sequencing on an Illumina MiSeq platform. The generated FASTQ files were aligned and scrutinized for each human platelet antigen polymorphism using SureCall data analysis software. Forty-six samples were successfully genotyped for human platelet antigens 1 through 29bw, with an average per base coverage depth of 1144. Concordance with historical human platelet antigen genotypes was 100%. A putative novel mutation in Exon 10 of the integrin β-3 (ITGB3) gene from an unsolved case of fetal neonatal alloimmune thrombocytopenia was also detected. A next-generation sequencing-based method that can accurately define all known human platelet antigen polymorphisms was developed. With the ability to sequence up to 96 samples simultaneously, our HaloPlex design could be used for high-throughput human platelet antigen genotyping. This method is also applicable for investigating fetal neonatal alloimmune thrombocytopenia when rare or novel human platelet antigens are suspected. © 2017 AABB.

Interval Between Infections and Viral Hierarchy Are Determinants of Viral Interference Following Influenza Virus Infection in a Ferret Model

PubMed Central

Laurie, Karen L.; Guarnaccia, Teagan A.; Carolan, Louise A.; Yan, Ada W. C.; Aban, Malet; Petrie, Stephen; Cao, Pengxing; Heffernan, Jane M.; McVernon, Jodie; Mosse, Jennifer; Kelso, Anne; McCaw, James M.; Barr, Ian G.

2015-01-01

Background. Epidemiological studies suggest that, following infection with influenza virus, there is a short period during which a host experiences a lower susceptibility to infection with other influenza viruses. This viral interference appears to be independent of any antigenic similarities between the viruses. We used the ferret model of human influenza to systematically investigate viral interference. Methods. Ferrets were first infected then challenged 1–14 days later with pairs of influenza A(H1N1)pdm09, influenza A(H3N2), and influenza B viruses circulating in 2009 and 2010. Results. Viral interference was observed when the interval between initiation of primary infection and subsequent challenge was <1 week. This effect was virus specific and occurred between antigenically related and unrelated viruses. Coinfections occurred when 1 or 3 days separated infections. Ongoing shedding from the primary virus infection was associated with viral interference after the secondary challenge. Conclusions. The interval between infections and the sequential combination of viruses were important determinants of viral interference. The influenza viruses in this study appear to have an ordered hierarchy according to their ability to block or delay infection, which may contribute to the dominance of different viruses often seen in an influenza season. PMID:25943206

The Viral Transcription Group Determines the HLA Class I Cellular Immune Response Against Human Respiratory Syncytial Virus*

PubMed Central

Johnstone, Carolina; Lorente, Elena; Barriga, Alejandro; Barnea, Eilon; Infantes, Susana; Lemonnier, François A.; David, Chella S.; Admon, Arie; López, Daniel

2015-01-01

The cytotoxic T-lymphocyte-mediated killing of virus-infected cells requires previous recognition of short viral antigenic peptides bound to human leukocyte antigen class I molecules that are exposed on the surface of infected cells. The cytotoxic T-lymphocyte response is critical for the clearance of human respiratory syncytial virus infection. In this study, naturally processed viral human leukocyte antigen class I ligands were identified with mass spectrometry analysis of complex human leukocyte antigen-bound peptide pools isolated from large amounts of human respiratory syncytial virus-infected cells. Acute antiviral T-cell response characterization showed that viral transcription determines both the immunoprevalence and immunodominance of the human leukocyte antigen class I response to human respiratory syncytial virus. These findings have clear implications for antiviral vaccine design. PMID:25635267

Viral Replication Complexes Are Targeted by LC3-Guided Interferon-Inducible GTPases.

PubMed

Biering, Scott B; Choi, Jayoung; Halstrom, Rachel A; Brown, Hailey M; Beatty, Wandy L; Lee, Sanghyun; McCune, Broc T; Dominici, Erin; Williams, Lelia E; Orchard, Robert C; Wilen, Craig B; Yamamoto, Masahiro; Coers, Jörn; Taylor, Gregory A; Hwang, Seungmin

2017-07-12

All viruses with positive-sense RNA genomes replicate on membranous structures in the cytoplasm called replication complexes (RCs). RCs provide an advantageous microenvironment for viral replication, but it is unknown how the host immune system counteracts these structures. Here we show that interferon-gamma (IFNG) disrupts the RC of murine norovirus (MNV) via evolutionarily conserved autophagy proteins and the induction of IFN-inducible GTPases, which are known to destroy the membrane of vacuoles containing bacteria, protists, or fungi. The MNV RC was marked by the microtubule-associated-protein-1-light-chain-3 (LC3) conjugation system of autophagy and then targeted by immunity-related GTPases (IRGs) and guanylate-binding proteins (GBPs) upon their induction by IFNG. Further, the LC3 conjugation system and the IFN-inducible GTPases were necessary to inhibit MNV replication in mice and human cells. These data suggest that viral RCs can be marked and antagonized by a universal immune defense mechanism targeting diverse pathogens replicating in cytosolic membrane structures. Copyright © 2017 Elsevier Inc. All rights reserved.

Vaccination and the TAP-independent antigen processing pathways.

PubMed

López, Daniel; Lorente, Elena; Barriga, Alejandro; Johnstone, Carolina; Mir, Carmen

2013-09-01

The cytotoxic CD8(+) T lymphocyte-mediated cellular response is important for the elimination of virus-infected cells and requires the prior recognition of short viral peptide antigens previously translocated to the endoplasmic reticulum by the transporter associated with antigen processing (TAP). However, individuals with nonfunctional TAP complexes or infected cells with TAP molecules blocked by specific viral proteins, such as the cowpoxvirus, a component of the first source of early empirical vaccination against smallpox, are still able to present several HLA class I ligands generated by the TAP-independent antigen processing pathways to specific cytotoxic CD8(+) T lymphocytes. Currently, bioterrorism and emerging infectious diseases have renewed interest in poxviruses. Recent works that have identified HLA class I ligands and epitopes in virus-infected TAP-deficient cells have implications for the study of both the effectiveness of early empirical vaccination and the analysis of HLA class I antigen processing in TAP-deficient subjects.

Preclinical Assessment of CAR T-Cell Therapy Targeting the Tumor Antigen 5T4 in Ovarian Cancer

PubMed Central

Owens, Gemma L.; Sheard, Victoria E.; Kalaitsidou, Milena; Blount, Daniel; Lad, Yatish; Cheadle, Eleanor J.; Edmondson, Richard J.; Kooner, Gurdeep; Gilham, David E.

2018-01-01

Chimeric antigen receptor (CAR) T cells represent a novel targeted approach to overcome both quantitative and qualitative shortfalls of the host immune system relating to the detection and subsequent destruction of tumors. The identification of antigens expressed specifically on the surface of tumor cells is a critical first step in the ability to utilize CAR T cells for the treatment of cancer. The 5T4 is a tumor-associated antigen which is expressed on the cell surface of most solid tumors including ovarian cancer. Matched blood and tumor samples were collected from 12 patients with ovarian cancer; all tumors were positive for 5T4 expression by immunohistochemistry. Patient T cells were effectively transduced with 2 different anti-5T4 CAR constructs which differed in their affinity for the target antigen. Co-culture of CAR T cells with matched autologous tumor disaggregates resulted in antigen-specific secretion of IFN-gamma. Furthermore, assessment of the efficacy of anti-5T4 CAR T cells in a mouse model resulted in therapeutic benefit against established ovarian tumors. These results demonstrate proof of principle that 5T4 is an attractive target for immune intervention in ovarian cancer and that patient T cells engineered to express a 5T4-specific CAR can recognize and respond physiologically to autologous tumor cells. PMID:29239915

High-throughput identification of antigen-specific TCRs by TCR gene capture.

PubMed

Linnemann, Carsten; Heemskerk, Bianca; Kvistborg, Pia; Kluin, Roelof J C; Bolotin, Dmitriy A; Chen, Xiaojing; Bresser, Kaspar; Nieuwland, Marja; Schotte, Remko; Michels, Samira; Gomez-Eerland, Raquel; Jahn, Lorenz; Hombrink, Pleun; Legrand, Nicolas; Shu, Chengyi Jenny; Mamedov, Ilgar Z; Velds, Arno; Blank, Christian U; Haanen, John B A G; Turchaninova, Maria A; Kerkhoven, Ron M; Spits, Hergen; Hadrup, Sine Reker; Heemskerk, Mirjam H M; Blankenstein, Thomas; Chudakov, Dmitriy M; Bendle, Gavin M; Schumacher, Ton N M

2013-11-01

The transfer of T cell receptor (TCR) genes into patient T cells is a promising approach for the treatment of both viral infections and cancer. Although efficient methods exist to identify antibodies for the treatment of these diseases, comparable strategies to identify TCRs have been lacking. We have developed a high-throughput DNA-based strategy to identify TCR sequences by the capture and sequencing of genomic DNA fragments encoding the TCR genes. We establish the value of this approach by assembling a large library of cancer germline tumor antigen-reactive TCRs. Furthermore, by exploiting the quantitative nature of TCR gene capture, we show the feasibility of identifying antigen-specific TCRs in oligoclonal T cell populations from either human material or TCR-humanized mice. Finally, we demonstrate the ability to identify tumor-reactive TCRs within intratumoral T cell subsets without knowledge of antigen specificities, which may be the first step toward the development of autologous TCR gene therapy to target patient-specific neoantigens in human cancer.

Species specific inhibition of viral replication using dicer substrate siRNAs (DsiRNAs) targeting the viral nucleoprotein of the fish pathogenic rhabdovirus viral hemorrhagic septicemia virus (VHSV).

PubMed

Bohle, Harry; Lorenzen, Niels; Schyth, Brian Dall

2011-06-01

Gene knock down by the use of small interfering RNAs (siRNAs) is widely used as a method for reducing the expression of specific genes in eukaryotic cells via the RNA interference pathway. But, the effectivity of siRNA induced gene knock down in cells from fish has in several studies been questioned and the specificity seems to be a general problem in cells originating from both lower and higher vertebrates. Here we show that we are able to reduce the level of viral gene expression and replication specifically in fish cells in vitro. We do so by using 27/25-mer DsiRNAs acting as substrates for dicer for the generation of siRNAs targeting the nucleoprotein N gene of viral hemorrhagic septicemia virus (VHSV). This rhabdovirus infects salmonid fish and is responsible for large yearly losses in aquaculture production. Specificity of the DsiRNA is assured in two ways: first, by using the conventional method of testing a control DsiRNA which should not target the gene of interest. Second, by assuring that replication of a heterologous virus of the same genus as the target virus was not inhibited by the DsiRNA. Target controls are, as we have previously highlighted, essential for verification of the specificity of siRNA-induced interference with virus multiplication, but they are still not in general use. Copyright © 2011 Elsevier B.V. All rights reserved.

Targeted delivery of antigen to hamster nasal lymphoid tissue with M-cell-directed lectins.

PubMed Central

Giannasca, P J; Boden, J A; Monath, T P

1997-01-01

The nasal cavity of a rodent is lined by an epithelium organized into distinct regional domains responsible for specific physiological functions. Aggregates of nasal lymphoid tissue (NALT) located at the base of the nasal cavity are believed to be sites of induction of mucosal immune responses to airborne antigens. The epithelium overlying NALT contains M cells which are specialized for the transcytosis of immunogens, as demonstrated in other mucosal tissues. We hypothesized that NALT M cells are characterized by distinct glycoconjugate receptors which influence antigen uptake and immune responses to transcytosed antigens. To identify glycoconjugates that may distinguish NALT M cells from other cells of the respiratory epithelium (RE), we performed lectin histochemistry on sections of the hamster nasal cavity with a panel of lectins. Many classes of glycoconjugates were found on epithelial cells in this region. While most lectins bound to sites on both the RE and M cells, probes capable of recognizing alpha-linked galactose were found to label the follicle-associated epithelium (FAE) almost exclusively. By morphological criteria, the FAE contains >90% M cells. To determine if apical glycoconjugates on M cells were accessible from the nasal cavity, an M-cell-selective lectin and a control lectin in parallel were administered intranasally to hamsters. The M-cell-selective lectin was found to specifically target the FAE, while the control lectin did not. Lectin bound to M cells in vivo was efficiently endocytosed, consistent with the role of M cells in antigen transport. Intranasal immunization with lectin-test antigen conjugates without adjuvant stimulated induction of specific serum immunoglobulin G, whereas antigen alone or admixed with lectin did not. The selective recognition of NALT M cells by a lectin in vivo provides a model for microbial adhesin-host cell receptor interactions on M cells and the targeted delivery of immunogens to NALT following intranasal

Assessment of cancer and virus antigens for cross-reactivity in human tissues.

PubMed

Jaravine, Victor; Raffegerst, Silke; Schendel, Dolores J; Frishman, Dmitrij

2017-01-01

Cross-reactivity (CR) or invocation of autoimmune side effects in various tissues has important safety implications in adoptive immunotherapy directed against selected antigens. The ability to predict CR (on-target and off-target toxicities) may help in the early selection of safer therapeutically relevant target antigens. We developed a methodology for the calculation of quantitative CR for any defined peptide epitope. Using this approach, we performed assessment of 4 groups of 283 currently known human MHC-class-I epitopes including differentiation antigens, overexpressed proteins, cancer-testis antigens and mutations displayed by tumor cells. In addition, 89 epitopes originating from viral sources were investigated. The natural occurrence of these epitopes in human tissues was assessed based on proteomics abundance data, while the probability of their presentation by MHC-class-I molecules was modelled by the method of Keşmir et al. which combines proteasomal cleavage, TAP affinity and MHC-binding predictions. The results of these analyses for many previously defined peptides are presented as CR indices and tissue profiles. The methodology thus allows for quantitative comparisons of epitopes and is suggested to be suited for the assessment of epitopes of candidate antigens in an early stage of development of adoptive immunotherapy. Our method is implemented as a Java program, with curated datasets stored in a MySQL database. It predicts all naturally possible self-antigens for a given sequence of a therapeutic antigen (or epitope) and after filtering for predicted immunogenicity outputs results as an index and profile of CR to the self-antigens in 22 human tissues. The program is implemented as part of the iCrossR webserver, which is publicly available at http://webclu.bio.wzw.tum.de/icrossr/ CONTACT: d.frishman@wzw.tum.deSupplementary information: Supplementary data are available at Bioinformatics online. © The Author 2016. Published by Oxford University Press

Antigenic Relationships Among Four Herpesviruses

PubMed Central

Blue, W. T.; Plummer, G.

1973-01-01

Common viral antigens were detected, by fluorescent-antibody studies, in cells infected with herpes simplex virus 1, squirrel monkey herpesvirus 1, bovine rhinotracheitis, and equine abortion viruses. The two primate viruses showed slight cross-neutralization. PMID:4351969

The viral transcription group determines the HLA class I cellular immune response against human respiratory syncytial virus.

PubMed

Johnstone, Carolina; Lorente, Elena; Barriga, Alejandro; Barnea, Eilon; Infantes, Susana; Lemonnier, François A; David, Chella S; Admon, Arie; López, Daniel

2015-04-01

The cytotoxic T-lymphocyte-mediated killing of virus-infected cells requires previous recognition of short viral antigenic peptides bound to human leukocyte antigen class I molecules that are exposed on the surface of infected cells. The cytotoxic T-lymphocyte response is critical for the clearance of human respiratory syncytial virus infection. In this study, naturally processed viral human leukocyte antigen class I ligands were identified with mass spectrometry analysis of complex human leukocyte antigen-bound peptide pools isolated from large amounts of human respiratory syncytial virus-infected cells. Acute antiviral T-cell response characterization showed that viral transcription determines both the immunoprevalence and immunodominance of the human leukocyte antigen class I response to human respiratory syncytial virus. These findings have clear implications for antiviral vaccine design. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

DNA vaccines targeting the encoded antigens to dendritic cells induce potent antitumor immunity in mice.

PubMed

Cao, Jun; Jin, Yiqi; Li, Wei; Zhang, Bin; He, Yang; Liu, Hongqiang; Xia, Ning; Wei, Huafeng; Yan, Jian

2013-08-14

Although DNA vaccine holds a great potential for cancer immunotherapy, effective long-lasting antitumoral immunity sufficient to induce durable responses in cancer patients remains to be achieved. Considering the pivotal role of dendritic cells (DC) in the antigen processing and presentation, we prepared DC-targeting DNA vaccines by fusing tumor-associated antigen HER2/neu ectodomain to single chain antibody fragment (scFv) from NLDC-145 antibody specific for DC-restricted surface molecule DEC-205 (scFvNLDC-145), and explored its antitumoral efficacy and underlying mechanisms in mouse breast cancer models. In vivo targeting assay demonstrated that scFvNLDC-145 specifically delivered DNA vaccine-encoded antigen to DC. Compared with untargeted HER2/neu DNA vaccines, vaccination with scFvNLDC-145-HER2/neu markedly promoted the HER2/neu-specific cellular and humoral immune responses with long-lasting immune memory, resulting in effective protection against challenge of HER2/neu-positive D2F2/E2 breast tumor while ineffective in parental HER2/neu-negative D2F2 breast tumor. More importantly, in combination with temporary depletion of regulatory T cells (Treg) by low-dose cyclophosphamide, vaccination with scFvNLDC-145-HER2/neu induced the regression of established D2F2/E2 breast tumor and significantly retarded the development of spontaneous mammary carcinomas in transgenic BALB-neuT mice. Our findings demonstrate that DC-targeted DNA vaccines for in vivo direct delivery of tumor antigens to DC could induce potent antigen-specific cellular and humoral immune responses and, if additional combination with systemic Treg depletion, was able to elicit an impressively therapeutic antitumoral activity, providing a rationale for further development of this approach for cancer treatment.

Tuning B cell responses to antigens by cell polarity and membrane trafficking.

PubMed

Del Valle Batalla, Felipe; Lennon-Dumenil, Ana-María; Yuseff, María-Isabel

2018-06-20

The capacity of B lymphocytes to produce specific antibodies, particularly broadly neutralizing antibodies that provide immunity to viral pathogens has positioned them as valuable therapeutic targets for immunomodulation. To become competent as antibody secreting cells, B cells undergo a series of activation steps, which are triggered by the recognition of antigens frequently displayed on the surface of other presenting cells. Such antigens elicit the formation of an immune synapse (IS), where local cytoskeleton rearrangements coupled to mechanical forces and membrane trafficking orchestrate the extraction and processing of antigens in B cells. In this review, we discuss the molecular mechanisms that regulate polarized membrane trafficking and mechanical properties of the immune synapse, as well as the potential extracellular cues from the environment, which may impact the ability of B cells to sense and acquire antigens at the immune synapse. An integrated view of the diverse cellular mechanisms that shape the immune synapse will provide a better understanding on how B cells are efficiently activated. Copyright © 2018 Elsevier Ltd. All rights reserved.

Optoacoustic sensing of ocular bacterial antigen using targeted gold nanorods

NASA Astrophysics Data System (ADS)

Maswadi, Saher; Page, Leland; Woodward, Lee; Glickman, Randolph D.; Barsalou, Norman

2008-02-01

Bacterial contamination can be detected using a minimally invasive optical method, based on laser-induced optoacoustic spectroscopy, to probe for specific antigens associated with a specific infectious agent. As a model system, we have used a surface antigen (Ag), isolated from Chlamydia trachomatis, and a complementary antibody (Ab). A preparation of 0.2 mg/ml of monoclonal Ab specific to the C. trachomatis surface Ag was conjugated to gold nanorods using standard commercial reagents, in order to produce a targeted contrast agent with a strong optoacoustic signal. The C. trachomatis Ag was absorbed in standard plastic microwells, and the binding of the complementary Ab-nanorod conjugate was tested in an immunoaffinity assay. Optoacoustic signals were elicited from the bound nanorods, using an optical parametric oscillator (OPO) laser system as the optical pump. The wavelength tuneability of the OPO optimized the spectroscopic measurement by exciting the nanorods at their optical absorption maxima. Optoacoustic responses were measured in the microwells using a probe beam deflection technique. Immunoaffinity assays were performed on several dilutions of purified C. trachomatis antigen ranging from 50 μg/ml to 1 pg/ml, in order to determine the detection limit for the optoacoustic-based assay. Only when the antigen was present, and the complementary Ab-NR reagent was introduced into the microwell, was an enhanced optoacoustic signal obtained, which indicated specific binding of the Ab-NR complex. The limit of detection with the current system design is between 1 and 5 pg/ml of bacterial Ag.

Quantitative immunophenotypic analysis of antigen-presenting cells involved in ectromelia virus antigen presentation in BALB/c and C57BL/6 mice.

PubMed

Szulc-Dąbrowska, Lidia; Gieryńska, Małgorzata; Boratyńska-Jasińska, Anna; Martyniszyn, Lech; Winnicka, Anna; Niemiałtowski, Marek G

2013-08-01

During mousepox in resistant (C57BL/6) or susceptible (BALB/c) strains of mice, stimulation of Th1 or Th2 cytokine immune response, respectively, is observed. Because mechanisms of different polarization of T cells remain elusive, in this study, we quantitatively assessed the phenotype of antigen-presenting cells (APCs) involved in ectromelia virus (ECTV) antigen presentation and cluster formation with effector cells in secondary lymphoid organs of BALB/c and C57BL/6 mice. We showed that both strains of mice display similar dynamics and kinetics of viral antigen presentation by CD11c(+) , CD11b(+) , and CD19(+) cells. CD11c(+) and CD11b(+) cells highly participated in viral antigen presentation during all stages of mousepox, whereas CD19(+) cells presented viral peptides later in infection. The main population of dendritic cells (DCs) engaged in ECTV antigen presentation and cell junction formation with effector cells was a population of myeloid CD11b(+) DCs (mDCs). We suggest that, on the one hand, ECTV may differentially affect the functions of APCs depending on the strain of mice. On the other hand, we suggest that some types of APCs, such as mDCs or other DCs subsets, have different abilities to direct the shape of immune response depending on the host resistance to mousepox. © 2013 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved.

Comparative evaluation of the diagnostic potential of recombinant envelope proteins and native cell culture purified viral antigens of Chikungunya virus.

PubMed

Khan, Mohsin; Dhanwani, Rekha; Kumar, Jyoti S; Rao, P V Lakshmana; Parida, Manmohan

2014-07-01

Despite the fact that Chikungunya resurgence is associated with epidemic of unprecedented magnitude, there are challenges in the field of its clinical diagnosis. However, serological tests in an ELISA format provide a rapid tool for the diagnosis of Chikungunya infection. Indeed, ELISAs based on recombinant proteins hold a great promise as these methods are cost effective and are free from the risk of handling biohazardous material. In this study, the performance of recombinant CHIKV antigens was compared in various ELISA formats for the diagnosis of Chikungunya. Two recombinant antigens derived from the envelope proteins of Chikungunya virus were prepared and evaluated by comparing their competence for detecting circulating antibodies in serum samples of patients infected with CHIKV using MAC-ELISA and indirect IgM-ELISA. The efficacy of the recombinant antigens was also compared with the native antigen. The indirect antibody capture IgM microplate ELISA revealed ≥90% concordance with the native antigen in detecting the CHIKV specific IgM antibodies whereas the recombinant antigen based MAC-ELISA showed 100% specificity. The recombinant antigens used in this study were effective and reliable targets for the diagnosis of CHIKV infection and also provide an alternative for native antigen use which is potentially biohazardous. © 2013 Wiley Periodicals, Inc.

RB1 is the crucial target of the Merkel cell polyomavirus Large T antigen in Merkel cell carcinoma cells.

PubMed

Hesbacher, Sonja; Pfitzer, Lisa; Wiedorfer, Katharina; Angermeyer, Sabrina; Borst, Andreas; Haferkamp, Sebastian; Scholz, Claus-Jürgen; Wobser, Marion; Schrama, David; Houben, Roland

2016-05-31

The pocket protein (PP) family consists of the three members RB1, p107 and p130 all possessing tumor suppressive properties. Indeed, the PPs jointly control the G1/S transition mainly by inhibiting E2F transcription factors. Notably, several viral oncoproteins are capable of binding and inhibiting PPs. Merkel cell polyomavirus (MCPyV) is considered as etiological factor for Merkel cell carcinoma (MCC) with expression of the viral Large T antigen (LT) harboring an intact PP binding domain being required for proliferation of most MCC cells. Therefore, we analyzed the interaction of MCPyV-LT with the PPs. Co-IP experiments indicate that MCPyV-LT binds potently only to RB1. Moreover, MCPyV-LT knockdown-induced growth arrest in MCC cells can be rescued by knockdown of RB1, but not by p107 or p130 knockdown. Accordingly, cell cycle arrest and E2F target gene repression mediated by the single PPs can only in the case of RB1 be significantly reverted by MCPyV-LT expression. Moreover, data from an MCC patient indicate that loss of RB1 rendered the MCPyV-positive MCC cells LT independent. Thus, our results suggest that RB1 is the dominant tumor suppressor PP in MCC, and that inactivation of RB1 by MCPyV-LT is largely sufficient for its growth supporting function in established MCPyV-positive MCC cells.

Metabolism Goes Viral

PubMed Central

Miyake-Stoner, Shigeki J.; O’Shea, Clodagh C.

2014-01-01

Viral and cellular oncogenes converge in targeting critical protein interaction networks to reprogram the cellular DNA and protein replication machinery for pathological replication. In this issue, Thai et al. (2014) show that adenovirus E4ORF1 activates MYC glycolytic targets to induce a Warburg-like effect that converts glucose into nucleotides for viral replication. PMID:24703688

Applying Convergent Immunity to Innovative Vaccines Targeting Staphylococcus aureus

PubMed Central

Yeaman, Michael R.; Filler, Scott G.; Schmidt, Clint S.; Ibrahim, Ashraf S.; Edwards, John E.; Hennessey, John P.

2014-01-01

Recent perspectives forecast a new paradigm for future “third generation” vaccines based on commonalities found in diverse pathogens or convergent immune defenses to such pathogens. For Staphylococcus aureus, recurring infections and a limited success of vaccines containing S. aureus antigens imply that native antigens induce immune responses insufficient for optimal efficacy. These perspectives exemplify the need to apply novel vaccine strategies to high-priority pathogens. One such approach can be termed convergent immunity, where antigens from non-target organisms that contain epitope homologs found in the target organism are applied in vaccines. This approach aims to evoke atypical immune defenses via synergistic processes that (1) afford protective efficacy; (2) target an epitope from one organism that contributes to protective immunity against another; (3) cross-protect against multiple pathogens occupying a common anatomic or immunological niche; and/or (4) overcome immune subversion or avoidance strategies of target pathogens. Thus, convergent immunity has a potential to promote protective efficacy not usually elicited by native antigens from a target pathogen. Variations of this concept have been mainstays in the history of viral and bacterial vaccine development. A more far-reaching example is the pre-clinical evidence that specific fungal antigens can induce cross-kingdom protection against bacterial pathogens. This trans-kingdom protection has been demonstrated in pre-clinical studies of the recombinant Candida albicans agglutinin-like sequence 3 protein (rAls3) where it was shown that a vaccine containing rAls3 provides homologous protection against C. albicans, heterologous protection against several other Candida species, and convergent protection against several strains of S. aureus. Convergent immunity reflects an intriguing new approach to designing and developing vaccine antigens and is considered here in the context of vaccines to target S

Sequence- and Interactome-Based Prediction of Viral Protein Hotspots Targeting Host Proteins: A Case Study for HIV Nef

PubMed Central

Sarmady, Mahdi; Dampier, William; Tozeren, Aydin

2011-01-01

Virus proteins alter protein pathways of the host toward the synthesis of viral particles by breaking and making edges via binding to host proteins. In this study, we developed a computational approach to predict viral sequence hotspots for binding to host proteins based on sequences of viral and host proteins and literature-curated virus-host protein interactome data. We use a motif discovery algorithm repeatedly on collections of sequences of viral proteins and immediate binding partners of their host targets and choose only those motifs that are conserved on viral sequences and highly statistically enriched among binding partners of virus protein targeted host proteins. Our results match experimental data on binding sites of Nef to host proteins such as MAPK1, VAV1, LCK, HCK, HLA-A, CD4, FYN, and GNB2L1 with high statistical significance but is a poor predictor of Nef binding sites on highly flexible, hoop-like regions. Predicted hotspots recapture CD8 cell epitopes of HIV Nef highlighting their importance in modulating virus-host interactions. Host proteins potentially targeted or outcompeted by Nef appear crowding the T cell receptor, natural killer cell mediated cytotoxicity, and neurotrophin signaling pathways. Scanning of HIV Nef motifs on multiple alignments of hepatitis C protein NS5A produces results consistent with literature, indicating the potential value of the hotspot discovery in advancing our understanding of virus-host crosstalk. PMID:21738584

Original antigenic sin responses to influenza viruses.

PubMed

Kim, Jin Hyang; Skountzou, Ioanna; Compans, Richard; Jacob, Joshy

2009-09-01

Most immune responses follow Burnet's rule in that Ag recruits specific lymphocytes from a large repertoire and induces them to proliferate and differentiate into effector cells. However, the phenomenon of "original antigenic sin" stands out as a paradox to Burnet's rule of B cell engagement. Humans, upon infection with a novel influenza strain, produce Abs against older viral strains at the expense of responses to novel, protective antigenic determinants. This exacerbates the severity of the current infection. This blind spot of the immune system and the redirection of responses to the "original Ag" rather than to novel epitopes were described fifty years ago. Recent reports have questioned the existence of this phenomenon. Hence, we revisited this issue to determine the extent to which original antigenic sin is induced by variant influenza viruses. Using two related strains of influenza A virus, we show that original antigenic sin leads to a significant decrease in development of protective immunity and recall responses to the second virus. In addition, we show that sequential infection of mice with two live influenza virus strains leads to almost exclusive Ab responses to the first viral strain, suggesting that original antigenic sin could be a potential strategy by which variant influenza viruses subvert the immune system.

Targeted gene delivery to the synovial pannus in antigen-induced arthritis by ultrasound-targeted microbubble destruction in vivo.

PubMed

Xiang, Xi; Tang, Yuanjiao; Leng, Qianying; Zhang, Lingyan; Qiu, Li

2016-02-01

The purpose of this study was to optimize an ultrasound-targeted microbubble destruction (UTMD) technique to improve the in vivo transfection efficiency of the gene encoding enhanced green fluorescent protein (EGFP) in the synovial pannus in an antigen-induced arthritis rabbit model. A mixture of microbubbles and plasmids was locally injected into the knee joints of an antigen-induced arthritis (AIA) rabbits. The plasmid concentrations and ultrasound conditions were varied in the experiments. We also tested local articular and intravenous injections. The rabbits were divided into five groups: (1) ultrasound+microbubbles+plasmid; (2) ultrasound+plasmid; (3) microbubble+plasmid; (4) plasmid only; (5) untreated controls. EGFP expression was observed by fluorescent microscope and immunohistochemical staining in the synovial pannus of each group. The optimal plasmid dosage and ultrasound parameter were determined based on the results of EGFP expression and the present and absent of tissue damage under light microscopy. The irradiation procedure was performed to observe the duration of the EGFP expression in the synovial pannus and other tissues and organs, as well as the damage to the normal cells. The optimal condition was determined to be a 1-MHz ultrasound pulse applied for 5 min with a power output of 2 W/cm(2) and a 20% duty cycle along with 300 μg of plasmid. Under these conditions, the synovial pannus showed significant EGFP expression without significant damage to the surrounding normal tissue. The EGFP expression induced by the local intra-articular injection was significantly more increased than that induced by the intravenous injection. The EGFP expression in the synovial pannus of the ultrasound+microbubbles+plasmid group was significantly higher than that of the other four groups (P<0.05). The expression peaked on day 5, remained detectable on day 40 and disappeared on day 60. No EGFP expression was detected in the other tissues and organs. The UTMD

Mannosylated poly(beta-amino esters) for targeted antigen presenting cell immune modulation

PubMed Central

Jones, Charles H.; Chen, Mingfu; Ravikrishnan, Anitha; Reddinger, Ryan; Zhang, Guojian; Hakansson, Anders P.; Pfeifer, Blaine A.

2014-01-01

Given the rise of antibiotic resistance and other difficult-to-treat diseases, genetic vaccination is a promising preventative approach that can be tailored and scaled according to the vector chosen for gene delivery. However, most vectors currently utilized rely on ubiquitous delivery mechanisms that ineffectively target important immune effectors such as antigen presenting cells (APCs). As such, APC targeting allows the option for tuning the direction (humoral vs cell-mediated) and strength of the resulting immune responses. In this work, we present the development and assessment of a library of mannosylated poly(beta-amino esters) (PBAEs) that represent a new class of easily synthesized APC-targeting cationic polymers. Polymeric characterization and assessment methodologies were designed to provide a more realistic physiochemical profile prior to in vivo evaluation. Gene delivery assessment in vitro showed significant improvement upon PBAE mannosylation and suggested that mannose-mediated uptake and processing influence the magnitude of gene delivery. Furthermore, mannosylated PBAEs demonstrated a strong, efficient, and safe in vivo humoral immune response without use of adjuvants when compared to genetic and protein control antigens. In summary, the gene delivery effectiveness provided by mannosylated PBAE vectors offers specificity and potency in directing APC activation and subsequent immune responses. PMID:25453962

Comparison of Detection Limits of 4th Generation Combination HIV Antigen/Antibody, p24 Antigen and Viral Load Assays on Diverse HIV Isolates.

PubMed

Stone, Mars; Bainbridge, John; Sanchez, Ana M; Keating, Sheila M; Pappas, Andrea; Rountree, Wes; Todd, Chris; Bakkour, Sonia; Manak, Mark; Peel, Sheila A; Coombs, Robert W; Ramos, Eric M; Shriver, M Kathleen; Contestable, Paul; Nair, Sangeetha Vijaysri; Wilson, David H; Stengelin, Martin; Murphy, Gary; Hewlett, Indira; Denny, Thomas N; Busch, Michael P

2018-05-23

Detection of acute HIV infection is critical for HIV public health and diagnostics. Clinical 4 th generation antigen-antibody (Ag/Ab) combination (combo) and p24 Ag immunoassays have enhanced detection of acute infection compared to Ab alone assays, but require ongoing evaluation with currently circulating diverse subtypes. Genetically and geographically diverse HIV clinical isolates were used to assess clinical HIV diagnostic, blood screening and next generation assays. Blinded 300 member panels of 20 serially diluted well-characterized antibody negative HIV isolates were distributed to manufacturers and end-user labs to assess relative analytic sensitivity of currently approved and pre-approved clinical HIV 4 th generation Ag/Ab combo or p24 Ag alone immunoassays across diverse subtypes. The limits of virus detection (LODs) were estimated for different subtypes relative to confirmed viral loads. Analysis of immunoassay sensitivity was benchmarked against confirmed viral load measurements on the blinded panel. Based on the proportion of positive results on 300 observations all Ag/Ab combo and standard sensitivity p24 Ag assays performed similarly and within half log LODs, illustrating similar breadth of reactivity and diagnostic utility. Ultrasensitive p24 Ag assays achieved dramatically increased sensitivities, while the rapid combo-assays performed poorly. Similar performance of the different commercially available 4 th gen assays on diverse subtypes supports their use in broad geographic settings with locally circulating HIV clades and recombinant strains. Next generation pre-clinical ultrasensitive p24 Ag assays achieved dramatically improved sensitivity, while p24 Ag detection by rapid 4 th gen assays performed poorly. Copyright © 2018 American Society for Microbiology.

Targeting HIV Reservoir in Infected CD4 T Cells by Dual-Affinity Re-targeting Molecules (DARTs) that Bind HIV Envelope and Recruit Cytotoxic T Cells

PubMed Central

Sloan, Derek D.; Lam, Chia-Ying Kao; Irrinki, Alivelu; Liu, Liqin; Tsai, Angela; Pace, Craig S.; Kaur, Jasmine; Murry, Jeffrey P.; Balakrishnan, Mini; Moore, Paul A.; Johnson, Syd; Nordstrom, Jeffrey L.; Cihlar, Tomas; Koenig, Scott

2015-01-01

HIV reservoirs and production of viral antigens are not eliminated in chronically infected participants treated with combination antiretroviral therapy (cART). Novel therapeutic strategies aiming at viral reservoir elimination are needed to address chronic immune dysfunction and non-AIDS morbidities that exist despite effective cART. The HIV envelope protein (Env) is emerging as a highly specific viral target for therapeutic elimination of the persistent HIV-infected reservoirs via antibody-mediated cell killing. Dual-Affinity Re-Targeting (DART) molecules exhibit a distinct mechanism of action via binding the cell surface target antigen and simultaneously engaging CD3 on cytotoxic T lymphocytes (CTLs). We designed and evaluated Env-specific DARTs (HIVxCD3 DARTs) derived from known antibodies recognizing diverse Env epitopes with or without broadly neutralizing activity. HIVxCD3 DARTs derived from PGT121, PGT145, A32, and 7B2, but not VRC01 or 10E8 antibodies, mediated potent CTL-dependent killing of quiescent primary CD4 T cells infected with diverse HIV isolates. Similar killing activity was also observed with DARTs structurally modified for in vivo half-life extension. In an ex vivo model using cells isolated from HIV-infected participants on cART, combinations of the most potent HIVxCD3 DARTs reduced HIV expression both in quiescent and activated peripheral blood mononuclear cell cultures isolated from HIV-infected participants on suppressive cART. Importantly, HIVxCD3 DARTs did not induce cell-to-cell virus spread in resting or activated CD4 T cell cultures. Collectively, these results provide support for further development of HIVxCD3 DARTs as a promising therapeutic strategy for targeting HIV reservoirs. PMID:26539983

MicroRNA-like viral small RNA from porcine reproductive and respiratory syndrome virus negatively regulates viral replication by targeting the viral nonstructural protein 2.

PubMed

Li, Na; Yan, Yunhuan; Zhang, Angke; Gao, Jiming; Zhang, Chong; Wang, Xue; Hou, Gaopeng; Zhang, Gaiping; Jia, Jinbu; Zhou, En-Min; Xiao, Shuqi

2016-12-13

Many viruses encode microRNAs (miRNAs) that are small non-coding single-stranded RNAs which play critical roles in virus-host interactions. Porcine reproductive and respiratory syndrome virus (PRRSV) is one of the most economically impactful viruses in the swine industry. The present study sought to determine whether PRRSV encodes miRNAs that could regulate PRRSV replication. Four viral small RNAs (vsRNAs) were mapped to the stem-loop structures in the ORF1a, ORF1b and GP2a regions of the PRRSV genome by bioinformatics prediction and experimental verification. Of these, the structures with the lowest minimum free energy (MFE) values predicted for PRRSV-vsRNA1 corresponded to typical stem-loop, hairpin structures. Inhibition of PRRSV-vsRNA1 function led to significant increases in viral replication. Transfection with PRRSV-vsRNA1 mimics significantly inhibited PRRSV replication in primary porcine alveolar macrophages (PAMs). The time-dependent increase in the abundance of PRRSV-vsRNA1 mirrored the gradual upregulation of PRRSV RNA expression. Knockdown of proteins associated with cellular miRNA biogenesis demonstrated that Drosha and Argonaute (Ago2) are involved in PRRSV-vsRNA1 biogenesis. Moreover, PRRSV-vsRNA1 bound specifically to the nonstructural protein 2 (NSP2)-coding sequence of PRRSV genome RNA. Collectively, the results reveal that PRRSV encodes a functional PRRSV-vsRNA1 which auto-regulates PRRSV replication by directly targeting and suppressing viral NSP2 gene expression. These findings not only provide new insights into the mechanism of the pathogenesis of PRRSV, but also explore a potential avenue for controlling PRRSV infection using viral small RNAs.

Molecular pathogenesis of viral hemorrhagic fever.

PubMed

Basler, Christopher F

2017-07-01

The clinical syndrome referred to as viral hemorrhagic fever (VHF) can be caused by several different families of RNA viruses, including select members of the arenaviruses, bunyaviruses, filoviruses, and flaviviruses. VHF is characterized by malaise, fever, vascular permeability, decreased plasma volume, coagulation abnormalities, and varying degrees of hemorrhage. Study of the filovirus Ebola virus has demonstrated a critical role for suppression of innate antiviral defenses in viral pathogenesis. Additionally, antigen-presenting cells are targets of productive infection and immune dysregulation. Among these cell populations, monocytes and macrophages are proposed to produce damaging inflammatory cytokines, while infected dendritic cells fail to undergo proper maturation, potentially impairing adaptive immunity. Uncontrolled virus replication and accompanying inflammatory responses are thought to promote vascular leakage and coagulopathy. However, the specific molecular pathways that underlie these features of VHF remain poorly understood. The arenavirus Lassa virus and the flavivirus yellow fever virus exhibit similar molecular pathogenesis suggesting common underlying mechanisms. Because non-human primate models that closely mimic VHF are available for Ebola, Lassa, and yellow fever viruses, we propose that comparative molecular studies using these models will yield new insights into the molecular underpinnings of VHF and suggest new therapeutic approaches.

Mannan-decorated thiolated Eudragit microspheres for targeting antigen presenting cells via nasal vaccination.

PubMed

Li, Hui-Shan; Singh, Bijay; Park, Tae-Eun; Hong, Zhong-Shan; Kang, Sang-Kee; Cho, Chong-Su; Choi, Yun-Jaie

2015-12-01

Mucosal vaccination of protein as an antigen requires appropriate delivery or adjuvant systems to deliver antigen to mucosal immune cells efficiently and generate valid immune responses. For successful nasal immunization, the obstacles imposed by the normal process of mucociliary clearance which limits residence time of applied antigens and low antigen delivery to antigen presenting cells (APCs) in nasal associated lymphoid tissue (NALT) need to be overcome for the efficient vaccination. Here, we prepared mucoadhesive and mannan-decorated thiolated Eudragit microspheres (Man-TEM) as a nasal vaccine carrier to overcome the limitations. Mucoadhesive thiolated Eudragit (TE) were decorated with mannan for targeting mannose receptors (MR) in antigen presenting cells (APCs) to obtain efficient immune responses. The potential adjuvant ability of Man-TEM for intranasal immunization was confirmed by in vitro and in vivo experiments. In mechanistic study using APCs in vitro, we obtained that Man-TEM enhanced the receptor-mediated endocytosis by stimulating the MR receptors of APCs. The nasal vaccination of OVA-loaded Man-TEM in mice showed higher levels of serum IgG and mucosal sIgA than the soluble OVA group due to the specific recognition of MR of APCs by the mannan in the Man-TEM. These results suggest that mucoadhesive and Man-TEM may be a promising candidate for nasal vaccine delivery system to elicit systemic and mucosal immunity. Copyright © 2015 Elsevier B.V. All rights reserved.

Hepatitis B virus core antigen determines viral persistence in a C57BL/6 mouse model.

PubMed

Lin, Yi-Jiun; Huang, Li-Rung; Yang, Hung-Chih; Tzeng, Horng-Tay; Hsu, Ping-Ning; Wu, Hui-Lin; Chen, Pei-Jer; Chen, Ding-Shinn

2010-05-18

We recently developed a mouse model of hepatitis B virus (HBV) persistence, in which a single i.v. hydrodynamic injection of HBV DNA to C57BL/6 mice allows HBV replication and induces a partial immune response, so that about 20-30% of the mice carry HBV for more than 6 months. The model was used to identify the viral antigen crucial for HBV persistence. We knocked out individual HBV genes by introducing a premature termination codon to the HBV core, HBeAg, HBx, and polymerase ORFs. The specific-gene-deficient HBV mutants were hydrodynamically injected into mice and the HBV profiles of the mice were monitored. About 90% of the mice that received the HBcAg-mutated HBV plasmid exhibited high levels of hepatitis B surface antigenemia and maintained HBsAg expression for more than 6 months after injection. To map the region of HBcAg essential for viral clearance, we constructed a set of serial HBcAg deletion mutants for hydrodynamic injection. We localized the essential region of HBcAg to the carboxyl terminus, specifically to the 10 terminal amino acids (HBcAg176-185). The majority of mice receiving this HBV mutant DNA did not elicit a proper HBcAg-specific IFN-gamma response and expressed HBV virions for 6 months. These results indicate that the immune response triggered in mice by HBcAg during exposure to HBV is important in determining HBV persistence.

Metabolism goes viral.

PubMed

Miyake-Stoner, Shigeki J; O'Shea, Clodagh C

2014-04-01

Viral and cellular oncogenes converge in targeting critical protein interaction networks to reprogram the cellular DNA and protein replication machinery for pathological replication. In this issue, Thai et al. (2014) show that adenovirus E4ORF1 activates MYC glycolytic targets to induce a Warburg-like effect that converts glucose into nucleotides for viral replication. Copyright © 2014 Elsevier Inc. All rights reserved.

Prostate-specific membrane antigen for prostate cancer theranostics: from imaging to targeted therapy.

PubMed

Arsenault, Frédéric; Beauregard, Jean-Mathieu; Pouliot, Frédéric

2018-06-22

In recent years, major advances in molecular imaging of prostate cancers (PCa) were made with the development and clinical validation of highly accurate PET tracers to stage and restage the disease. Prostate-specific membrane antigen (PSMA) is a transmembrane protein highly expressed in PCa, and its expression has led to the development of PSMA-binding radiopharmaceuticals for molecular imaging or radioligand therapy (RLT). We herein review the recent literature published on diagnostic and therapeutic (i.e. theranostic) PSMA tracers. Development in small PSMA-targeted molecules labeled with gallium-68 and fluorine-18 show promising results for primary staging and detection of disease at biochemical recurrence using PET/computed tomography (PET/CT). Studies show a higher sensitivity and specificity, along with an improved detection rate over conventional imaging (CT scan and bone scan) or choline PET tracers, especially for restaging after prostate-specific antigen failure following loco-regional therapy. In addition, some PSMA tracers can be labeled with beta-minus and alpha particle emitters, yielding encouraging response rates and low toxicity, and potentially offering a new line of targeted therapy for metastatic castration-resistant PCa. PSMA-targeted tracers have shown unprecedented accuracy to stage and restage PCa using PET/CT. Given their specific biodistribution toward PCa tissue, PSMA RLT now offers new therapeutic possibilities to target metastatic PCa. Prospective multicenter randomized studies investigating the clinical impact management impacts of PSMA-targeted molecules are urgently needed.

Engineered Lentivector Targeting of Dendritic Cells for In Vivo Immunization

PubMed Central

Yang, Lili; Yang, Haiguang; Rideout, Kendra; Cho, Taehoon; Joo, Kye il; Ziegler, Leslie; Elliot, Abigail; Walls, Anthony; Yu, Dongzi; Baltimore, David; Wang, Pin

2008-01-01

We report a method of inducing antigen production in dendritic cells (DCs) by in vivo targeting with lentiviral vectors that specifically bind to the DC surface protein, DC-SIGN. To target the DCs, the lentivector was enveloped with a viral glycoprotein from Sindbis virus, engineered to be DC-SIGN-specific. In vitro, this lentivector specifically transduced DCs and induced DC maturation. A remarkable frequency (up to 12%) of ovalbumin (OVA)-specific CD8+ T cells and a significant antibody response were observed 2 weeks following injection of a targeted lentiviral vector encoding an OVA transgene into naïve mice. These mice were solidly protected against the growth of the OVA-expressing E.G7 tumor and this methodology could even induce regression of an established tumor. Thus, lentiviral vectors targeting DCs provide a simple method of producing effective immunity and may provide an alternative route for immunization with protein antigens. PMID:18297056

Antigen Cross-Priming of Cell-Associated Proteins is Enhanced by Macroautophagy within the Antigen Donor Cell

PubMed Central

Joubert, Pierre-Emmanuel; Albert, Matthew L.

2012-01-01

Phagocytosis of dying cells constitutes an important mechanism of antigen capture for the cross-priming of CD8+ T cells. This process has been shown to be critical for achieving tumor and viral immunity. While most studies have focused on the mechanisms inherent in the dendritic cell that account for exogenous antigen accessing MHC I, several recent reports have highlighted the important contribution made by the antigen donor cell. Specifically, the cell stress and cell death pathways that precede antigen transfer are now known to impact cross-presentation and cross-priming. Herein, we review the current literature regarding a role for macroautophagy within the antigen donor cell. Further examination of this point of immune regulation is warranted and may contribute to a better understanding of how to optimize immunotherapy for treatment of cancer and chronic infectious disease. PMID:22566942

Lentiviral Protein Transfer Vectors Are an Efficient Vaccine Platform and Induce a Strong Antigen-Specific Cytotoxic T Cell Response

PubMed Central

Uhlig, Katharina M.; Schülke, Stefan; Scheuplein, Vivian A. M.; Malczyk, Anna H.; Reusch, Johannes; Kugelmann, Stefanie; Muth, Anke; Koch, Vivian; Hutzler, Stefan; Bodmer, Bianca S.; Schambach, Axel; Buchholz, Christian J.; Waibler, Zoe; Scheurer, Stephan

2015-01-01

ABSTRACT To induce and trigger innate and adaptive immune responses, antigen-presenting cells (APCs) take up and process antigens. Retroviral particles are capable of transferring not only genetic information but also foreign cargo proteins when they are genetically fused to viral structural proteins. Here, we demonstrate the capacity of lentiviral protein transfer vectors (PTVs) for targeted antigen transfer directly into APCs and thereby induction of cytotoxic T cell responses. Targeting of lentiviral PTVs to APCs can be achieved analogously to gene transfer vectors by pseudotyping the particles with truncated wild-type measles virus (MV) glycoproteins (GPs), which use human SLAM (signaling lymphocyte activation molecule) as a main entry receptor. SLAM is expressed on stimulated lymphocytes and APCs, including dendritic cells. SLAM-targeted PTVs transferred the reporter protein green fluorescent protein (GFP) or Cre recombinase with strict receptor specificity into SLAM-expressing CHO and B cell lines, in contrast to broadly transducing vesicular stomatitis virus G protein (VSV-G) pseudotyped PTVs. Primary myeloid dendritic cells (mDCs) incubated with targeted or nontargeted ovalbumin (Ova)-transferring PTVs stimulated Ova-specific T lymphocytes, especially CD8+ T cells. Administration of Ova-PTVs into SLAM-transgenic and control mice confirmed the observed predominant induction of antigen-specific CD8+ T cells and demonstrated the capacity of protein transfer vectors as suitable vaccines for the induction of antigen-specific immune responses. IMPORTANCE This study demonstrates the specificity and efficacy of antigen transfer by SLAM-targeted and nontargeted lentiviral protein transfer vectors into antigen-presenting cells to trigger antigen-specific immune responses in vitro and in vivo. The observed predominant activation of antigen-specific CD8+ T cells indicates the suitability of SLAM-targeted and also nontargeted PTVs as a vaccine for the induction of

Strategies to alleviate original antigenic sin responses to influenza viruses.

PubMed

Kim, Jin Hyang; Davis, William G; Sambhara, Suryaprakash; Jacob, Joshy

2012-08-21

Original antigenic sin is a phenomenon wherein sequential exposure to closely related influenza virus variants reduces antibody (Ab) response to novel antigenic determinants in the second strain and, consequently, impairs the development of immune memory. This could pose a risk to the development of immune memory in persons previously infected with or vaccinated against influenza. Here, we explored strategies to overcome original antigenic sin responses in mice sequentially exposed to two closely related hemagglutinin 1 neuraminidase 1 (H1N1) influenza strains A/PR/8/34 and A/FM/1/47. We found that dendritic cell-activating adjuvants [Bordetella pertussis toxin (PT) or CpG ODN or a squalene-based oil-in-water nanoemulsion (NE)], upon administration during the second viral exposure, completely protected mice from a lethal challenge and enhanced neutralizing-Ab titers against the second virus. Interestingly, PT and NE adjuvants when administered during the first immunization even prevented original antigenic sin in subsequent immunization without any adjuvants. As an alternative to using adjuvants, we also found that repeated immunization with the second viral strain relieved the effects of original antigenic sin. Taken together, our studies provide at least three ways of overcoming original antigenic sin.

Prostate-specific membrane antigen-directed nanoparticle targeting for extreme nearfield ablation of prostate cancer cells.

PubMed

Lee, Seung S; Roche, Philip Jr; Giannopoulos, Paresa N; Mitmaker, Elliot J; Tamilia, Michael; Paliouras, Miltiadis; Trifiro, Mark A

2017-03-01

Almost all biological therapeutic interventions cannot overcome neoplastic heterogeneity. Physical ablation therapy is immune to tumor heterogeneity, but nearby tissue damage is the limiting factor in delivering lethal doses. Multi-walled carbon nanotubes offer a number of unique properties: chemical stability, photonic properties including efficient light absorption, thermal conductivity, and extensive surface area availability for covalent chemical ligation. When combined together with a targeting moiety such as an antibody or small molecule, one can deliver highly localized temperature increases and cause extensive cellular damage. We have functionalized multi-walled carbon nanotubes by conjugating an antibody against prostate-specific membrane antigen. In our in vitro studies using prostate-specific membrane antigen-positive LNCaP prostate cancer cells, we have effectively demonstrated cell ablation of >80% with a single 30-s exposure to a 2.7-W, 532-nm laser for the first time without bulk heating. We also confirmed the specificity and selectivity of prostate-specific membrane antigen targeting by assessing prostate-specific membrane antigen-null PC3 cell lines under the same conditions (<10% cell ablation). This suggests that we can achieve an extreme nearfield cell ablation effect, thus restricting potential tissue damage when transferred to in vivo clinical applications. Developing this new platform will introduce novel approaches toward current therapeutic modalities and will usher in a new age of effective cancer treatment squarely addressing tumoral heterogeneity.

T cells for viral infections after allogeneic hematopoietic stem cell transplant

PubMed Central

Heslop, Helen E.

2016-01-01

Despite recent advances in the field of allogeneic hematopoietic stem cell transplantation (HSCT), viral infections are still a major complication during the period of immune suppression that follows the procedure. Adoptive transfer of donor-derived virus-specific cytotoxic T cells (VSTs) is a strategy to rapidly restore virus-specific immunity to prevent or treat viral diseases after HSCT. Early proof of principle studies demonstrated that the administration of donor-derived T cells specific for cytomegalovirus or Epstein-Barr virus (EBV) could effectively restore virus-specific immunity and control viral infections. Subsequent studies using different expansion or direct selection techniques have shown that donor-derived VSTs confer protection in vivo after adoptive transfer in 70% to 90% of recipients. Because a major cause of failure is lack of immunity to the infecting virus in a naïve donor, more recent studies have infused closely matched third-party VSTs and reported response rates of 60% to 70%. Current efforts have focused on broadening the applicability of this approach by: (1) extending the number of viral antigens being targeted, (2) simplifying manufacture, (3) exploring strategies for recipients of virus-naïve donor grafts, and (4) developing and optimizing “off the shelf” approaches. PMID:27207801

T cells for viral infections after allogeneic hematopoietic stem cell transplant.

PubMed

Bollard, Catherine M; Heslop, Helen E

2016-06-30

Despite recent advances in the field of allogeneic hematopoietic stem cell transplantation (HSCT), viral infections are still a major complication during the period of immune suppression that follows the procedure. Adoptive transfer of donor-derived virus-specific cytotoxic T cells (VSTs) is a strategy to rapidly restore virus-specific immunity to prevent or treat viral diseases after HSCT. Early proof of principle studies demonstrated that the administration of donor-derived T cells specific for cytomegalovirus or Epstein-Barr virus (EBV) could effectively restore virus-specific immunity and control viral infections. Subsequent studies using different expansion or direct selection techniques have shown that donor-derived VSTs confer protection in vivo after adoptive transfer in 70% to 90% of recipients. Because a major cause of failure is lack of immunity to the infecting virus in a naïve donor, more recent studies have infused closely matched third-party VSTs and reported response rates of 60% to 70%. Current efforts have focused on broadening the applicability of this approach by: (1) extending the number of viral antigens being targeted, (2) simplifying manufacture, (3) exploring strategies for recipients of virus-naïve donor grafts, and (4) developing and optimizing "off the shelf" approaches. © 2016 by The American Society of Hematology.

Role of Metalloproteases in Vaccinia Virus Epitope Processing for Transporter Associated with Antigen Processing (TAP)-independent Human Leukocyte Antigen (HLA)-B7 Class I Antigen Presentation*

PubMed Central

Lorente, Elena; García, Ruth; Mir, Carmen; Barriga, Alejandro; Lemonnier, François A.; Ramos, Manuel; López, Daniel

2012-01-01

The transporter associated with antigen processing (TAP) translocates the viral proteolytic peptides generated by the proteasome and other proteases in the cytosol to the endoplasmic reticulum lumen. There, they complex with nascent human leukocyte antigen (HLA) class I molecules, which are subsequently recognized by the CD8+ lymphocyte cellular response. However, individuals with nonfunctional TAP complexes or tumor or infected cells with blocked TAP molecules are able to present HLA class I ligands generated by TAP-independent processing pathways. Herein, using a TAP-independent polyclonal vaccinia virus-polyspecific CD8+ T cell line, two conserved vaccinia-derived TAP-independent HLA-B*0702 epitopes were identified. The presentation of these epitopes in normal cells occurs via complex antigen-processing pathways involving the proteasome and/or different subsets of metalloproteinases (amino-, carboxy-, and endoproteases), which were blocked in infected cells with specific chemical inhibitors. These data support the hypothesis that the abundant cellular proteolytic systems contribute to the supply of peptides recognized by the antiviral cellular immune response, thereby facilitating immunosurveillance. These data may explain why TAP-deficient individuals live normal life spans without any increased susceptibility to viral infections. PMID:22298786

Development of a multicomponent vaccine for Streptococcus pyogenes based on the antigenic targets of IVIG.

PubMed

Reglinski, Mark; Lynskey, Nicola N; Choi, Yoon Jung; Edwards, Robert J; Sriskandan, Shiranee

2016-04-01

Despite over a century of research and the careful scrutiny of many promising targets, there is currently no vaccine available for the prevention of Streptococcus pyogenes infection. Through analysis of the protective, anti-streptococcal components of pooled human immunoglobulin, we previously identified ten highly conserved and invariant S. pyogenes antigens that contribute to anti-streptococcal immunity in the adult population. We sought to emulate population immunity to S. pyogenes through a process of active vaccination, using the antigens targeted by pooled human immunoglobulin. Seven targets were produced recombinantly and mixed to form a multicomponent vaccine (Spy7). Vaccinated mice were challenged with S. pyogenes isolates representing four globally relevant serotypes (M1, M3, M12 and M89) using an established model of invasive disease. Vaccination with Spy7 stimulated the production of anti-streptococcal antibodies, and limited systemic dissemination of M1 and M3 S. pyogenes from an intramuscular infection focus. Vaccination additionally attenuated disease severity due to M1 S. pyogenes as evidenced by reduction in weight loss, and modulated cytokine release. Spy7 vaccination successfully stimulated the generation of protective anti-streptococcal immunity in vivo. Identification of reactive antigens using pooled human immunoglobulin may represent a novel route to vaccine discovery for extracellular bacteria. Copyright © 2016 The Authors. Published by Elsevier Ltd.. All rights reserved.

Development of a multicomponent vaccine for Streptococcus pyogenes based on the antigenic targets of IVIG

PubMed Central

Reglinski, Mark; Lynskey, Nicola N.; Choi, Yoon Jung; Edwards, Robert J.; Sriskandan, Shiranee

2016-01-01

Summary Objectives Despite over a century of research and the careful scrutiny of many promising targets, there is currently no vaccine available for the prevention of Streptococcus pyogenes infection. Through analysis of the protective, anti-streptococcal components of pooled human immunoglobulin, we previously identified ten highly conserved and invariant S. pyogenes antigens that contribute to anti-streptococcal immunity in the adult population. We sought to emulate population immunity to S. pyogenes through a process of active vaccination, using the antigens targeted by pooled human immunoglobulin. Methods Seven targets were produced recombinantly and mixed to form a multicomponent vaccine (Spy7). Vaccinated mice were challenged with S. pyogenes isolates representing four globally relevant serotypes (M1, M3, M12 and M89) using an established model of invasive disease. Results Vaccination with Spy7 stimulated the production of anti-streptococcal antibodies, and limited systemic dissemination of M1 and M3 S. pyogenes from an intramuscular infection focus. Vaccination additionally attenuated disease severity due to M1 S. pyogenes as evidenced by reduction in weight loss, and modulated cytokine release. Conclusion Spy7 vaccination successfully stimulated the generation of protective anti-streptococcal immunity in vivo. Identification of reactive antigens using pooled human immunoglobulin may represent a novel route to vaccine discovery for extracellular bacteria. PMID:26880087

Tumor target amplification: Implications for nano drug delivery systems.

PubMed

Seidi, Khaled; Neubauer, Heidi A; Moriggl, Richard; Jahanban-Esfahlan, Rana; Javaheri, Tahereh

2018-04-10

Tumor cells overexpress surface markers which are absent from normal cells. These tumor-restricted antigenic signatures are a fundamental basis for distinguishing on-target from off-target cells for ligand-directed targeting of cancer cells. Unfortunately, tumor heterogeneity impedes the establishment of a solid expression pattern for a given target marker, leading to drastic changes in quality (availability) and quantity (number) of the target. Consequently, a subset of cancer cells remains untargeted during the course of treatment, which subsequently promotes drug-resistance and cancer relapse. Since target inefficiency is only problematic for cancer treatment and not for treatment of other pathological conditions such as viral/bacterial infections, target amplification or the generation of novel targets is key to providing eligible antigenic markers for effective targeted therapy. This review summarizes the limitations of current ligand-directed targeting strategies and provides a comprehensive overview of tumor target amplification strategies, including self-amplifying systems, dual targeting, artificial markers and peptide modification. We also discuss the therapeutic and diagnostic potential of these approaches, the underlying mechanism(s) and established methodologies, mostly in the context of different nanodelivery systems, to facilitate more effective ligand-directed cancer cell monitoring and targeting. Copyright © 2018 Elsevier B.V. All rights reserved.

Influenza virus infection elicits protective antibodies and T cells specific for host cell antigens also expressed as tumor associated antigens: a new view of cancer immunosurveillance

PubMed Central

Iheagwara, Uzoma K.; Beatty, Pamela L.; Van, Phu T.; Ross, Ted M.; Minden, Jonathan S.; Finn, Olivera J.

2014-01-01

Most tumor-associated antigens (TAA) are self-molecules that are abnormally expressed in cancer cells and become targets of antitumor immune responses. Antibodies and T cells specific for some TAA have been found in healthy individuals and are associated with lowered lifetime risk for developing cancer. Lower risk for cancer has also been associated with a history of febrile viral diseases. We hypothesized that virus infections could lead to transient expression of abnormal forms of self-molecules, some of which are TAA; facilitated by the adjuvant effects of infection and inflammation, these molecules could elicit specific antibodies, T cells and lasting immune memory simultaneously with immunity against viral antigens. Such infection-induced immune memory for TAA would be expected to provide life-long immune surveillance of cancer. Using influenza virus infection in mice as a model system, we tested this hypothesis and demonstrated that influenza-experienced mice control 3LL mouse lung tumor challenge better than infection-naive control mice. Using 2D-Difference Gel Electrophoresis (2D-DIGE) and mass spectrometry, we identified numerous molecules, some of which are known TAA, on the 3LL tumor cells recognized by antibodies elicited by two successive influenza infections. We studied in detail immune responses against GAPDH, Histone H4, HSP90, Malate Dehydrogenase 2 and Annexin A2, all of which were overexpressed in influenza-infected lungs and in tumor cells. Lastly, we show that immune responses generated through vaccination against peptides derived from these antigens correlated with improved tumor control. PMID:24778322

A Bioinformatic Pipeline for Monitoring of the Mutational Stability of Viral Drug Targets with Deep-Sequencing Technology.

PubMed

Kravatsky, Yuri; Chechetkin, Vladimir; Fedoseeva, Daria; Gorbacheva, Maria; Kravatskaya, Galina; Kretova, Olga; Tchurikov, Nickolai

2017-11-23

The efficient development of antiviral drugs, including efficient antiviral small interfering RNAs (siRNAs), requires continuous monitoring of the strict correspondence between a drug and the related highly variable viral DNA/RNA target(s). Deep sequencing is able to provide an assessment of both the general target conservation and the frequency of particular mutations in the different target sites. The aim of this study was to develop a reliable bioinformatic pipeline for the analysis of millions of short, deep sequencing reads corresponding to selected highly variable viral sequences that are drug target(s). The suggested bioinformatic pipeline combines the available programs and the ad hoc scripts based on an original algorithm of the search for the conserved targets in the deep sequencing data. We also present the statistical criteria for the threshold of reliable mutation detection and for the assessment of variations between corresponding data sets. These criteria are robust against the possible sequencing errors in the reads. As an example, the bioinformatic pipeline is applied to the study of the conservation of RNA interference (RNAi) targets in human immunodeficiency virus 1 (HIV-1) subtype A. The developed pipeline is freely available to download at the website http://virmut.eimb.ru/. Brief comments and comparisons between VirMut and other pipelines are also presented.

Factors affecting the stability of viral vaccines.

PubMed

Peetermans, J

1996-01-01

The stability of viral vaccines is determined by the rate of loss of "integrity" of the viral antigen during storage. For live vaccines, such as measles, mumps, rubella, canine distemper, stability is equivalent to the preservation of the infectious titres. For inactivated and subunit vaccines, the preservation of the antigenic structure and the correct steric presentation of the relevant epitopes are the parameters which determine their stability. In general, the following factors may have a negative effect on stability: temperature, pH outside the physiological limits, organic solvents, repeated freezing and thawing, some antiseptics and inactivating agents, and light. However their negative effect is in most cases specific for the individual viruses. Approaches to stabilisation of most vaccines are based on the elimination or neutralisation of the negative factors. Practical examples for the most relevant existing vaccines are described.

Engineering HIV-Specific Immunity with Chimeric Antigen Receptors.

PubMed

Kitchen, Scott G; Zack, Jerome A

2016-12-01

HIV remains a highly important public health and clinical issue despite many recent advances in attempting to develop a cure, which has remained elusive for most people infected with HIV. HIV disease can be controlled with pharmacologic therapies; however, these treatments are expensive, may have severe side effects, and are not curative. Consequently, an improved means to control or eliminate HIV replication is needed. Cytotoxic T lymphocytes (CTLs) play a critical role in controlling viral replication and are an important part in the ability of the immune response to eradicate most viral infections. There are considerable efforts to enhance CTL responses in HIV-infected individuals in hopes of providing the immune response with armaments to more effectively control viral replication. In this review, we discuss some of these efforts and focus on the development of a gene therapy-based approach to engineer hematopoietic stem cells with an HIV-1-specific chimeric antigen receptor, which seeks to provide an inexhaustible source of HIV-1-specific immune cells that are MHC unrestricted and superior to natural antiviral T cell responses. These efforts provide the basis for further development of T cell functional enhancement to target and treat chronic HIV infection in hopes of eradicating the virus from the body.

Bat Caliciviruses and Human Noroviruses Are Antigenically Similar and Have Overlapping Histo-Blood Group Antigen Binding Profiles

PubMed Central

Lindesmith, Lisa C.; Debbink, Kari; Beall, Anne; Mallory, Michael L.; Yount, Boyd L.; Graham, Rachel L.; Huynh, Jeremy; Gates, J. Edward; Donaldson, Eric F.

2018-01-01

ABSTRACT Emerging zoonotic viral diseases remain a challenge to global public health. Recent surveillance studies have implicated bats as potential reservoirs for a number of viral pathogens, including coronaviruses and Ebola viruses. Caliciviridae represent a major viral family contributing to emerging diseases in both human and animal populations and have been recently identified in bats. In this study, we blended metagenomics, phylogenetics, homology modeling, and in vitro assays to characterize two novel bat calicivirus (BtCalV) capsid sequences, corresponding to strain BtCalV/A10/USA/2009, identified in Perimyotis subflavus near Little Orleans, MD, and bat norovirus. We observed that bat norovirus formed virus-like particles and had epitopes and receptor-binding patterns similar to those of human noroviruses. To determine whether these observations stretch across multiple bat caliciviruses, we characterized a novel bat calicivirus, BtCalV/A10/USA/2009. Phylogenetic analysis revealed that BtCalV/A10/USA/2009 likely represents a novel Caliciviridae genus and is most closely related to "recoviruses." Homology modeling revealed that the capsid sequences of BtCalV/A10/USA/2009 and bat norovirus resembled human norovirus capsid sequences and retained host ligand binding within the receptor-binding domains similar to that seen with human noroviruses. Both caliciviruses bound histo-blood group antigens in patterns that overlapped those seen with human and animal noroviruses. Taken together, our results indicate the potential for bat caliciviruses to bind histo-blood group antigens and overcome a significant barrier to cross-species transmission. Additionally, we have shown that bat norovirus maintains antigenic epitopes similar to those seen with human noroviruses, providing further evidence of evolutionary descent. Our results reiterate the importance of surveillance of wild-animal populations, especially of bats, for novel viral pathogens. PMID:29789360

Viral Perturbations of Host Networks Reflect Disease Etiology

PubMed Central

Dricot, Amélie; Padi, Megha; Byrdsong, Danielle; Franchi, Rachel; Lee, Deok-Sun; Rozenblatt-Rosen, Orit; Mar, Jessica C.; Calderwood, Michael A.; Baldwin, Amy; Zhao, Bo; Santhanam, Balaji; Braun, Pascal; Simonis, Nicolas; Huh, Kyung-Won; Hellner, Karin; Grace, Miranda; Chen, Alyce; Rubio, Renee; Marto, Jarrod A.; Christakis, Nicholas A.; Kieff, Elliott; Roth, Frederick P.; Roecklein-Canfield, Jennifer; DeCaprio, James A.; Cusick, Michael E.; Quackenbush, John; Hill, David E.; Münger, Karl; Vidal, Marc; Barabási, Albert-László

2012-01-01

Many human diseases, arising from mutations of disease susceptibility genes (genetic diseases), are also associated with viral infections (virally implicated diseases), either in a directly causal manner or by indirect associations. Here we examine whether viral perturbations of host interactome may underlie such virally implicated disease relationships. Using as models two different human viruses, Epstein-Barr virus (EBV) and human papillomavirus (HPV), we find that host targets of viral proteins reside in network proximity to products of disease susceptibility genes. Expression changes in virally implicated disease tissues and comorbidity patterns cluster significantly in the network vicinity of viral targets. The topological proximity found between cellular targets of viral proteins and disease genes was exploited to uncover a novel pathway linking HPV to Fanconi anemia. PMID:22761553

Antibody to the rheumatoid arthritis nuclear antigen. Its relationship to in vivo Epstein-Barr virus infection.

PubMed

Catalano, M A; Carson, D A; Niederman, J C; Feorino, P; Vaughan, J H

1980-05-01

Most patients with seropositive rheumatoid arthritis, and a variable but lesser percentage of normal subjects, have precipitating antibodies to a nuclear antigen, rheumatoid arthritis nuclear antigen, present in Epstein-Barr virus-infected human B lymphoblastoid cells. We have used a sensitive indirect immunofluorescence assay for antibody to rheumatoid arthritis nuclear antigen in a study of patients with infectious mononucleosis and healthy control subjects. Of 110 sera from normal, college-age cadets, 58 were from individuals without prior Epstein-Barr virus infection, as indicated by the lack of antibody to viral capsid antigen. All of these also lacked activity to rheumatoid arthritis nuclear antigen. 52 sera were positive for antibody to viral capsid antigen, and antibody to rheumatoid arthritis nuclear antigen was present in 26 (50%) of these. In 67 sequential sera from 11 college-age students with infectious mononucleosis who became positive for antibody to rheumatoid arthritis nuclear antigen, only 2 were positive during the 1 mo. Thereafter the incidence and titers increased progressively through the 1st yr after infection. This time-course resembled that for the development of antibody to Epstein-Barr nuclear antigen, another transformation antigen in Epstein-Barr virus-infected B lymphocytes. The development of positivity for both was much later than that of antibody to the structural viral capsid antigen, which in the current study was always positive by 1 wk. Thus, antibody to rheumatoid arthritis nuclear antigen is present in a large proportion of normal individuals and can now be clearly ascribed, from both in vivo and in vitro studies, to prior infection with Epstein-Barr virus.

Viral Evasion of Natural Killer Cell Activation

PubMed Central

Ma, Yi; Li, Xiaojuan; Kuang, Ersheng

2016-01-01

Natural killer (NK) cells play a key role in antiviral innate defenses because of their abilities to kill infected cells and secrete regulatory cytokines. Additionally, NK cells exhibit adaptive memory-like antigen-specific responses, which represent a novel antiviral NK cell defense mechanism. Viruses have evolved various strategies to evade the recognition and destruction by NK cells through the downregulation of the NK cell activating receptors. Here, we review the recent findings on viral evasion of NK cells via the impairment of NK cell-activating receptors and ligands, which provide new insights on the relationship between NK cells and viral actions during persistent viral infections. PMID:27077876

Viral Evasion of Natural Killer Cell Activation.

PubMed

Ma, Yi; Li, Xiaojuan; Kuang, Ersheng

2016-04-12

Natural killer (NK) cells play a key role in antiviral innate defenses because of their abilities to kill infected cells and secrete regulatory cytokines. Additionally, NK cells exhibit adaptive memory-like antigen-specific responses, which represent a novel antiviral NK cell defense mechanism. Viruses have evolved various strategies to evade the recognition and destruction by NK cells through the downregulation of the NK cell activating receptors. Here, we review the recent findings on viral evasion of NK cells via the impairment of NK cell-activating receptors and ligands, which provide new insights on the relationship between NK cells and viral actions during persistent viral infections.

Targeting Membrane-Bound Viral RNA Synthesis Reveals Potent Inhibition of Diverse Coronaviruses Including the Middle East Respiratory Syndrome Virus

PubMed Central

Bergström, Tomas; Kann, Nina; Adamiak, Beata; Hannoun, Charles; Kindler, Eveline; Jónsdóttir, Hulda R.; Muth, Doreen; Kint, Joeri; Forlenza, Maria; Müller, Marcel A.; Drosten, Christian; Thiel, Volker; Trybala, Edward

2014-01-01

Coronaviruses raise serious concerns as emerging zoonotic viruses without specific antiviral drugs available. Here we screened a collection of 16671 diverse compounds for anti-human coronavirus 229E activity and identified an inhibitor, designated K22, that specifically targets membrane-bound coronaviral RNA synthesis. K22 exerts most potent antiviral activity after virus entry during an early step of the viral life cycle. Specifically, the formation of double membrane vesicles (DMVs), a hallmark of coronavirus replication, was greatly impaired upon K22 treatment accompanied by near-complete inhibition of viral RNA synthesis. K22-resistant viruses contained substitutions in non-structural protein 6 (nsp6), a membrane-spanning integral component of the viral replication complex implicated in DMV formation, corroborating that K22 targets membrane bound viral RNA synthesis. Besides K22 resistance, the nsp6 mutants induced a reduced number of DMVs, displayed decreased specific infectivity, while RNA synthesis was not affected. Importantly, K22 inhibits a broad range of coronaviruses, including Middle East respiratory syndrome coronavirus (MERS–CoV), and efficient inhibition was achieved in primary human epithelia cultures representing the entry port of human coronavirus infection. Collectively, this study proposes an evolutionary conserved step in the life cycle of positive-stranded RNA viruses, the recruitment of cellular membranes for viral replication, as vulnerable and, most importantly, druggable target for antiviral intervention. We expect this mode of action to serve as a paradigm for the development of potent antiviral drugs to combat many animal and human virus infections. PMID:24874215

DNA vaccines against viral diseases of farmed fish.

PubMed

Evensen, Øystein; Leong, Jo-Ann C

2013-12-01

Immunization by an antigen-encoding DNA was approved for commercial sale in Canada against a Novirhabdovirus infection in fish. DNA vaccines have been particularly successful against the Novirhabdoviruses while there are reports on the efficacy against viral pathogens like infectious pancreatic necrosis virus, infectious salmon anemia virus, and lymphocystis disease virus and these are inferior to what has been attained for the novirhabdoviruses. Most recently, DNA vaccination of Penaeus monodon against white spot syndrome virus was reported. Research efforts are now focused on the development of more effective vectors for DNA vaccines, improvement of vaccine efficacy against various viral diseases of fish for which there is currently no vaccines available and provision of co-expression of viral antigen and immunomodulatory compounds. Scientists are also in the process of developing new delivery methods. While a DNA vaccine has been approved for commercial use in farmed salmon in Canada, it is foreseen that it is still a long way to go before a DNA vaccine is approved for use in farmed fish in Europe. Copyright © 2013 Elsevier Ltd. All rights reserved.

Rational design of nanoparticles towards targeting antigen-presenting cells and improved T cell priming.

PubMed

Zupančič, Eva; Curato, Caterina; Paisana, Maria; Rodrigues, Catarina; Porat, Ziv; Viana, Ana S; Afonso, Carlos A M; Pinto, João; Gaspar, Rogério; Moreira, João N; Satchi-Fainaro, Ronit; Jung, Steffen; Florindo, Helena F

2017-07-28

Vaccination is a promising strategy to trigger and boost immune responses against cancer or infectious disease. We have designed, synthesized and characterized aliphatic-polyester (poly(lactic-co-glycolic acid) (PLGA) nanoparticles (NP) to investigate how the nature of protein association (adsorbed versus entrapped) and polymer/surfactant concentrations impact on the generation and modulation of antigen-specific immune responses. The ability of the NP formulations to target dendritic cells (DC), be internalized and activate the T cells was characterized and optimized in vitro and in vivo using markers of DC activation and co-stimulatory molecules. Ovalbumin (OVA) was used as a model antigen in combination with the engraftment of CD4 + and CD8 + T cells, carrying a transgenic OVA-responding T cell receptor (TCR), to trace and characterize the activation of antigen-specific CD4 + and CD8 + lymph node T cells upon NP vaccination. Accordingly, the phenotype and frequency of immune cell stimulation induced by the NP loaded with OVA, isolated or in combination with synthetic unmethylated cytosine-phosphate-guanine (CpG) oligodeoxynucleotide (ODN) motifs, were characterized. DC-NP interactions increased with incubation time, presenting internalization values between 50 and 60% and 30-40%, in vitro and in vivo, respectively. Interestingly, animal immunization with antigen-adsorbed NP up-regulated major histocompatibility complex (MHC) class II (MHCII), while NP entrapping the antigen up-regulated MHCI, suggesting a more efficient cross-presentation. On the other hand, rather surprisingly, the surfactant used in the NP formulation had a major impact on the activation of antigen presenting cells (APC). In fact, DC collected from lymph nodes of animals immunized with NP prepared using poly(vinil alcohol) (PVA), as a surfactant, expressed significantly higher levels of CD86, MHCI and MHCII. In addition, those NP prepared with PVA and co-entrapping OVA and the toll

Antibodies against the mono-methylated arginine-glycine repeat (MMA-RG) of the Epstein-Barr virus nuclear antigen 2 (EBNA2) identify potential cellular proteins targeted in viral transformation.

PubMed

Ayoubian, Hiresh; Fröhlich, Thomas; Pogodski, Dagmar; Flatley, Andrew; Kremmer, Elisabeth; Schepers, Aloys; Feederle, Regina; Arnold, Georg J; Grässer, Friedrich A

2017-08-01

The Epstein-Barr virus is a human herpes virus with oncogenic potential. The virus-encoded nuclear antigen 2 (EBNA2) is a key mediator of viral tumorigenesis. EBNA2 features an arginine-glycine (RG) repeat at amino acids (aa)339-354 that is essential for the transformation of lymphocytes and contains symmetrically (SDMA) and asymmetrically (ADMA) di-methylated arginine residues. The SDMA-modified EBNA2 binds the survival motor neuron protein (SMN), thus mimicking SMD3, a cellular SDMA-containing protein that interacts with SMN. Accordingly, a monoclonal antibody (mAb) specific for the SDMA-modified RG repeat of EBNA2 also binds to SMD3. With the novel mAb 19D4 we now show that EBNA2 contains mono-methylated arginine (MMA) residues within the RG repeat. Using 19D4, we immune-precipitated and analysed by mass spectrometry cellular proteins in EBV-transformed B-cells that feature MMA motifs that are similar to the one in EBNA2. Among the cellular proteins identified, we confirmed by immunoprecipitation and/or Western blot analyses Aly/REF, Coilin, DDX5, FXR1, HNRNPK, LSM4, MRE11, NRIP, nucleolin, PRPF8, RBM26, SMD1 (SNRDP1) and THRAP3 proteins that are either known to contain MMA residues or feature RG repeat sequences that probably serve as methylation substrates. The identified proteins are involved in splicing, tumorigenesis, transcriptional activation, DNA stability and RNA processing or export. Furthermore, we found that several proteins involved in energy metabolism are associated with MMA-modified proteins. Interestingly, the viral EBNA1 protein that features methylated RG repeat motifs also reacted with the antibodies. Our results indicate that the region between aa 34-52 of EBNA1 contains ADMA or SDMA residues, while the region between aa 328-377 mainly contains MMA residues.

Targeting nanosystems to human DCs via Fc receptor as an effective strategy to deliver antigen for immunotherapy.

PubMed

Cruz, Luis J; Rueda, Felix; Cordobilla, Begoña; Simón, Lorena; Hosta, Leticia; Albericio, Fernando; Domingo, Joan Carles

2011-02-07

Dendritic cells (DCs) are increasingly being explored as cellular vaccines for tumor immunotherapy, since they provide an effective system of antigen presentation both in vitro and in vivo. An additional advantage of this cell type is that it is possible to target specific antigens through the activation of receptors, such as FcR (the receptor for the IgG Fc fragment) and TLR (toll-like Receptor). Thus, the uptake capacity of DCs can be improved, thereby increasing antigen presentation. This, in turn, would lead to an enhanced immune response, and, in some instances, the tolerance/anergy of immune effector cells present in cancer patients could be reverted. Here we studied various nanotargeting systems, including liposomes and gold nanoparticles of a peptide-based immunotherapeutic vaccine for the treatment of androgen-responsive prostate cancer. Building blocks of the immunogenic peptide consisted of the luteinizing hormone-releasing hormone (LHRH), also known as gonadotropin-releasing hormone (GnRH) peptide (B- and T-cell epitope), in tandem with a T-helper epitope corresponding to the 830-844 region of tetanus toxoid. Three new peptides with several modifications at the N-terminal (palmitoyl, acetyl, and FITC) were synthesized. These peptides also contained a Cys as C-terminal residue to facilitate grafting onto gold nanoparticles. To target different antigen formulations to human DCs, the Fc was activated with a cross-linking spacer to generate a free thiol group and thus facilitate conjugation onto gold nanoparticles, liposomes, and peptide. Our results show that gold nanoparticles and liposomes targeted to FcRs of human DCs are effective antigen delivery carriers and induce a strong immune response with respect to nontargeted LHRH-TT-nanoparticle conjugates and a superior response to that of naked antigens. In addition, dual labeling using gold and FITC-peptide allowed DC tracking by flow cytometry as well as transmission electron microscopy. Nanoparticles

Mutational analysis of polyomavirus small-T-antigen functions in productive infection and in transformation.

PubMed

Martens, I; Nilsson, S A; Linder, S; Magnusson, G

1989-05-01

The function of polyomavirus small T antigen in productive infection and in transformation was studied. Transfection of permissive mouse cells with mixtures of mutants that express only one type of T antigen showed that small T antigen increased large-T-antigen-dependent viral DNA synthesis approximately 10-fold. Under the same conditions, small T antigen was also essential for the formation of infectious virus particles. To analyze these activities of small T antigen, mutants producing protein with single amino acid replacements were constructed. Two mutants, bc1073 and bc1075, were characterized. Although both mutations led to the substitution of amino acid residues of more than one T antigen, the phenotype of both mutants was associated with alterations of the small T antigen. Both mutant proteins had lost their activity in the maturation of infectious virus particles. The bc1075 but not the bc1073 small T antigen had also lost its ability to stimulate viral DNA synthesis in mouse 3T6 cells. Finally, both mutants retained a third activity of small T antigen: to confer on rat cells also expressing middle T antigen the ability to grow efficiently in semisolid medium. The phenotypes of the mutants in these three assays suggest that small T antigen has at least three separate functions.

Antibodies elicited by the first non-viral prophylactic cancer vaccine show tumor-specificity and immunotherapeutic potential

PubMed Central

Lohmueller, Jason J.; Sato, Shuji; Popova, Lana; Chu, Isabel M.; Tucker, Meghan A.; Barberena, Roberto; Innocenti, Gregory M.; Cudic, Mare; Ham, James D.; Cheung, Wan Cheung; Polakiewicz, Roberto D.; Finn, Olivera J.

2016-01-01

MUC1 is a shared tumor antigen expressed on >80% of human cancers. We completed the first prophylactic cancer vaccine clinical trial based on a non-viral antigen, MUC1, in healthy individuals at-risk for colon cancer. This trial provided a unique source of potentially effective and safe immunotherapeutic drugs, fully-human antibodies affinity-matured in a healthy host to a tumor antigen. We purified, cloned, and characterized 13 IgGs specific for several tumor-associated MUC1 epitopes with a wide range of binding affinities. These antibodies bind hypoglycosylated MUC1 on human cancer cell lines and tumor tissues but show no reactivity against fully-glycosylated MUC1 on normal cells and tissues. We found that several antibodies activate complement-mediated cytotoxicity and that T cells carrying chimeric antigen receptors with the antibody variable regions kill MUC1+ target cells, express activation markers, and produce interferon gamma. Fully-human and tumor-specific, these antibodies are candidates for further testing and development as immunotherapeutic drugs. PMID:27545199

Integrating influenza antigenic dynamics with molecular evolution

PubMed Central

Bedford, Trevor; Suchard, Marc A; Lemey, Philippe; Dudas, Gytis; Gregory, Victoria; Hay, Alan J; McCauley, John W; Russell, Colin A; Smith, Derek J; Rambaut, Andrew

2014-01-01

Influenza viruses undergo continual antigenic evolution allowing mutant viruses to evade host immunity acquired to previous virus strains. Antigenic phenotype is often assessed through pairwise measurement of cross-reactivity between influenza strains using the hemagglutination inhibition (HI) assay. Here, we extend previous approaches to antigenic cartography, and simultaneously characterize antigenic and genetic evolution by modeling the diffusion of antigenic phenotype over a shared virus phylogeny. Using HI data from influenza lineages A/H3N2, A/H1N1, B/Victoria and B/Yamagata, we determine patterns of antigenic drift across viral lineages, showing that A/H3N2 evolves faster and in a more punctuated fashion than other influenza lineages. We also show that year-to-year antigenic drift appears to drive incidence patterns within each influenza lineage. This work makes possible substantial future advances in investigating the dynamics of influenza and other antigenically-variable pathogens by providing a model that intimately combines molecular and antigenic evolution. DOI: http://dx.doi.org/10.7554/eLife.01914.001 PMID:24497547

Specific Antibodies Reacting with SV40 Large T Antigen Mimotopes in Serum Samples of Healthy Subjects

PubMed Central

Tognon, Mauro; Corallini, Alfredo; Manfrini, Marco; Taronna, Angelo; Butel, Janet S.; Pietrobon, Silvia; Trevisiol, Lorenzo; Bononi, Ilaria; Vaccher, Emanuela; Barbanti-Brodano, Giuseppe; Martini, Fernanda; Mazzoni, Elisa

2016-01-01

Simian Virus 40, experimentally assayed in vitro in different animal and human cells and in vivo in rodents, was classified as a small DNA tumor virus. In previous studies, many groups identified Simian Virus 40 sequences in healthy individuals and cancer patients using PCR techniques, whereas others failed to detect the viral sequences in human specimens. These conflicting results prompted us to develop a novel indirect ELISA with synthetic peptides, mimicking Simian Virus 40 capsid viral protein antigens, named mimotopes. This immunologic assay allowed us to investigate the presence of serum antibodies against Simian Virus 40 and to verify whether Simian Virus 40 is circulating in humans. In this investigation two mimotopes from Simian Virus 40 large T antigen, the viral replication protein and oncoprotein, were employed to analyze for specific reactions to human sera antibodies. This indirect ELISA with synthetic peptides from Simian Virus 40 large T antigen was used to assay a new collection of serum samples from healthy subjects. This novel assay revealed that serum antibodies against Simian Virus 40 large T antigen mimotopes are detectable, at low titer, in healthy subjects aged from 18–65 years old. The overall prevalence of reactivity with the two Simian Virus 40 large T antigen peptides was 20%. This new ELISA with two mimotopes of the early viral regions is able to detect in a specific manner Simian Virus 40 large T antigen-antibody responses. PMID:26731525

Structure, sequence and expression of the hepatitis delta (δ) viral genome

NASA Astrophysics Data System (ADS)

Wang, Kang-Sheng; Choo, Qui-Lim; Weiner, Amy J.; Ou, Jing-Hsiung; Najarian, Richard C.; Thayer, Richard M.; Mullenbach, Guy T.; Denniston, Katherine J.; Gerin, John L.; Houghton, Michael

1986-10-01

Biochemical and electron microscopic data indicate that the human hepatitis δ viral agent contains a covalently closed circular and single-stranded RNA genome that has certain similarities with viroid-like agents from plants. The sequence of the viral genome (1,678 nucleotides) has been determined and an open reading frame within the complementary strand has been shown to encode an antigen that binds specifically to antisera from patients with chronic hepatitis δ viral infections.

Enterovirus 71 encephalomyelitis and Japanese encephalitis can be distinguished by topographic distribution of inflammation and specific intraneuronal detection of viral antigen and RNA.

PubMed

Wong, K T; Ng, K Y; Ong, K C; Ng, W F; Shankar, S K; Mahadevan, A; Radotra, B; Su, I J; Lau, G; Ling, A E; Chan, K P; Macorelles, P; Vallet, S; Cardosa, M J; Desai, A; Ravi, V; Nagata, N; Shimizu, H; Takasaki, T

2012-08-01

To investigate if two important epidemic viral encephalitis in children, Enterovirus 71 (EV71) encephalomyelitis and Japanese encephalitis (JE) whose clinical and pathological features may be nonspecific and overlapping, could be distinguished. Tissue sections from the central nervous system of infected cases were examined by light microscopy, immunohistochemistry and in situ hybridization. All 13 cases of EV71 encephalomyelitis collected from Asia and France invariably showed stereotyped distribution of inflammation in the spinal cord, brainstem, hypothalamus, cerebellar dentate nucleus and, to a lesser extent, cerebral cortex and meninges. Anterior pons, corpus striatum, thalamus, temporal lobe, hippocampus and cerebellar cortex were always uninflamed. In contrast, the eight JE cases studied showed inflammation involving most neuronal areas of the central nervous system, including the areas that were uninflamed in EV71 encephalomyelitis. Lesions in both infections were nonspecific, consisting of perivascular and parenchymal infiltration by inflammatory cells, oedematous/necrolytic areas, microglial nodules and neuronophagia. Viral inclusions were absent. Immunohistochemistry and in situ hybridization assays were useful to identify the causative virus, localizing viral antigens and RNA, respectively, almost exclusively to neurones. The stereotyped distribution of inflammatory lesions in EV71 encephalomyelitis appears to be very useful to help distinguish it from JE. © 2011 The Authors. Neuropathology and Applied Neurobiology © 2011 British Neuropathological Society.

Folate receptor 1 (FOLR1) targeted chimeric antigen receptor (CAR) T cells for the treatment of gastric cancer

PubMed Central

Pyo, Suhkneung; Kang, Chung Hyo; Lee, Chong Ock; Lee, Heung Kyoung; Choi, Sang Un; Park, Chi Hoon

2018-01-01

Gastric cancer is a malignancy that has a high mortality rate. Although progress has been made in the treatment of gastric cancer, many patients experience cancer recurrence and metastasis. Folate receptor 1 (FOLR1) is overexpressed on the cell surface in over one-third of gastric cancer patients, but rarely is expressed in normal tissue. This makes FOLR1 a potential target for chimeric antigen receptor (CAR) T cell immunotherapy, although the function of FOLR1 has not been elucidated. CAR are engineered fusion receptor composed of an antigen recognition region and signaling domains. T cells expressing CAR have specific activation and cytotoxic effects against cancer cells containing the target antigen. In this study, we generated a CAR that targets FOLR1 composed of a single-chain variable fragment (scFv) of FOLR1 antibody and signaling domains consisting of CD28 and CD3ζ. Both FOLR1-CAR KHYG-1, a natural killer cell line, and FOLR1-CAR T cells recognized FOLR1-positive gastric cancer cells in a MHC-independent manner and induced secretion of various cytokines and caused cell death. Conclusively, this is the first study to demonstrate that CAR KHYG-1/T cells targeting FOLR1 are effective against FOLR1-positive gastric cancer cells. PMID:29874279

Replicating viral vector platform exploits alarmin signals for potent CD8+ T cell-mediated tumour immunotherapy

PubMed Central

Kallert, Sandra M.; Darbre, Stephanie; Bonilla, Weldy V.; Kreutzfeldt, Mario; Page, Nicolas; Müller, Philipp; Kreuzaler, Matthias; Lu, Min; Favre, Stéphanie; Kreppel, Florian; Löhning, Max; Luther, Sanjiv A.; Zippelius, Alfred; Merkler, Doron; Pinschewer, Daniel D.

2017-01-01

Viral infections lead to alarmin release and elicit potent cytotoxic effector T lymphocyte (CTLeff) responses. Conversely, the induction of protective tumour-specific CTLeff and their recruitment into the tumour remain challenging tasks. Here we show that lymphocytic choriomeningitis virus (LCMV) can be engineered to serve as a replication competent, stably-attenuated immunotherapy vector (artLCMV). artLCMV delivers tumour-associated antigens to dendritic cells for efficient CTL priming. Unlike replication-deficient vectors, artLCMV targets also lymphoid tissue stroma cells expressing the alarmin interleukin-33. By triggering interleukin-33 signals, artLCMV elicits CTLeff responses of higher magnitude and functionality than those induced by replication-deficient vectors. Superior anti-tumour efficacy of artLCMV immunotherapy depends on interleukin-33 signalling, and a massive CTLeff influx triggers an inflammatory conversion of the tumour microenvironment. Our observations suggest that replicating viral delivery systems can release alarmins for improved anti-tumour efficacy. These mechanistic insights may outweigh safety concerns around replicating viral vectors in cancer immunotherapy. PMID:28548102

Challenge with Bovine viral diarrhea virus by exposure to persistently infected calves: protection by vaccination and negative results of antigen testing in nonvaccinated acutely infected calves

PubMed Central

Johnson, Bill J.; Briggs, Robert E.; Ridpath, Julia F.; Saliki, Jeremiah T.; Confer, Anthony W.; Burge, Lurinda J.; Step, Douglas L.; Walker, Derek A.; Payton, Mark E.

2006-01-01

Abstract Calves persistently infected (PI) with Bovine viral diarrhea virus (BVDV) represent an important source of infection for susceptible cattle. We evaluated vaccine efficacy using calves PI with noncytopathic BVDV2a for the challenge and compared tests to detect BVDV in acutely or transiently infected calves versus PI calves. Vaccination with 2 doses of modified live virus vaccine containing BVDV1a and BVDV2a protected the calves exposed to the PI calves: neither viremia nor nasal shedding occurred. An immunohistochemistry test on formalin-fixed ear notches and an antigen-capture enzyme-linked immunosorbent assay on fresh notches in phosphate-buffered saline did not detect BVDV antigen in any of the acutely or transiently infected calves, whereas both tests had positive results in all the PI calves. PMID:16639944

Structural and Nonstructural Viral Proteins Are Targets of T-Helper Immune Response against Human Respiratory Syncytial Virus.

PubMed

Lorente, Elena; Barriga, Alejandro; Barnea, Eilon; Mir, Carmen; Gebe, John A; Admon, Arie; López, Daniel

2016-06-01

Proper antiviral humoral and cellular immune responses require previous recognition of viral antigenic peptides that are bound to HLA class II molecules, which are exposed on the surface of antigen-presenting cells. The helper immune response is critical for the control and the clearance of human respiratory syncytial virus (HRSV) infection, a virus with severe health risk in infected pediatric, immunocompromised, and elderly populations. In this study, using a mass spectrometry analysis of complex HLA class II-bound peptide pools that were isolated from large amounts of HRSV-infected cells, 19 naturally processed HLA-DR ligands, most of them included in a complex nested set of peptides, were identified. Both the immunoprevalence and the immunodominance of the HLA class II response to HRSV were focused on one nonstructural (NS1) and two structural (matrix and mainly fusion) proteins of the infective virus. These findings have clear implications for analysis of the helper immune response as well as for antiviral vaccine design. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

Original antigenic sin: A comprehensive review.

PubMed

Vatti, Anup; Monsalve, Diana M; Pacheco, Yovana; Chang, Christopher; Anaya, Juan-Manuel; Gershwin, M Eric

2017-09-01

The concept of "original antigenic sin" was first proposed by Thomas Francis, Jr. in 1960. This phenomenon has the potential to rewrite what we understand about how the immune system responds to infections and its mechanistic implications on how vaccines should be designed. Antigenic sin has been demonstrated to occur in several infectious diseases in both animals and humans, including human influenza infection and dengue fever. The basis of "original antigenic sin" requires immunological memory, and our immune system ability to autocorrect. In the context of viral infections, it is expected that if we are exposed to a native strain of a pathogen, we should be able to mount a secondary immune response on subsequent exposure to the same pathogen. "Original antigenic sin" will not contradict this well-established immunological process, as long as the subsequent infectious antigen is identical to the original one. But "original antigenic sin" implies that when the epitope varies slightly, then the immune system relies on memory of the earlier infection, rather than mount another primary or secondary response to the new epitope which would allow faster and stronger responses. The result is that the immunological response may be inadequate against the new strain, because the immune system does not adapt and instead relies on its memory to mount a response. In the case of vaccines, if we only immunize to a single strain or epitope, and if that strain/epitope changes over time, then the immune system is unable to mount an accurate secondary response. In addition, depending of the first viral exposure the secondary immune response can result in an antibody-dependent enhancement of the disease or at the opposite, it could induce anergy. Both of them triggering loss of pathogen control and inducing aberrant clinical consequences. Copyright © 2017 Elsevier Ltd. All rights reserved.

Targeting the IL-33/IL-13 Axis for Respiratory Viral Infections.

PubMed

Donovan, Chantal; Bourke, Jane E; Vlahos, Ross

2016-04-01

Lung diseases, such as asthma and chronic obstructive pulmonary disease (COPD), are highly prevalent worldwide. One of the major factors that limits the efficacy of current medication in these patients are viral infections, leading to exacerbations of symptoms and decreased quality of life. Current pharmacological strategies targeting virus-induced lung disease are problematic due to antiviral resistance and the requirement for strain-specific vaccination. Thus, new therapeutic strategies are urgently required. In this Opinion article, we provide state-of-the-art evidence from humans and preclinical animal models implicating the interleukin (IL)-33/IL-13 axis in virus-induced lung disease. Thus, targeting the IL-33/IL-13 axis may be a feasible way to overcome the limitations of current therapy used to treat virus-induced exacerbations of lung disease. Copyright © 2016 Elsevier Ltd. All rights reserved.

Cellular sources and targets of IFN-gamma-mediated protection against viral demyelination and neurological deficits.

PubMed

Murray, Paul D; McGavern, Dorian B; Pease, Larry R; Rodriguez, Moses

2002-03-01

IFN-gamma is an anti-viral and immunomodulatory cytokine critical for resistance to multiple pathogens. Using mice with targeted disruption of the gene for IFN-gamma, we previously demonstrated that this cytokine is critical for resistance to viral persistence and demyelination in the Theiler's virus model of multiple sclerosis. During viral infections, IFN-gamma is produced by natural killer (NK) cells, CD4(+) and CD8(+) T cells; however, the proportions of lymphocyte subsets responding to virus infection influences the contributions to IFN-gamma-mediated protection. To determine the lymphocyte subsets that produce IFN-gamma to maintain resistance, we used adoptive transfer strategies to generate mice with lymphocyte-specific deficiencies in IFN-gamma-production. We demonstrate that IFN-gamma production by both CD4(+) and CD8(+) T cell subsets is critical for resistance to Theiler's murine encephalomyelitis virus (TMEV)-induced demyelination and neurological disease, and that CD4(+) T cells make a greater contribution to IFN-gamma-mediated protection. To determine the cellular targets of IFN-gamma-mediated responses, we used adoptive transfer studies and bone marrow chimerism to generate mice in which either hematopoietic or somatic cells lacked the ability to express IFN-gamma receptor. We demonstrate that IFN-gamma receptor must be present on central nervous system glia, but not bone marrow-derived lymphocytes, in order to maintain resistance to TMEV-induced demyelination.

Cellular sources and targets of IFN-γ-mediated protection against viral demyelination and neurological deficits

PubMed Central

Murray, Paul D.; McGavern, Dorian B.; Pease, Larry R.; Rodriguez, Moses

2017-01-01

IFN-γ is an anti-viral and immunomodulatory cytokine critical for resistance to multiple pathogens. Using mice with targeted disruption of the gene for IFN-γ, we previously demonstrated that this cytokine is critical for resistance to viral persistence and demyelination in the Theiler’s virus model of multiple sclerosis. During viral infections, IFN-γ is produced by natural killer (NK) cells, CD4+ and CD8+ T cells; however, the proportions of lymphocyte subsets responding to virus infection influences the contributions to IFN-γ-mediated protection. To determine the lymphocyte subsets that produce IFN-γ to maintain resistance, we used adoptive transfer strategies to generate mice with lymphocyte-specific deficiencies in IFN-γ-production. We demonstrate that IFN-γ production by both CD4+ and CD8+ T cell subsets is critical for resistance to Theiler’s murine encephalomyelitis virus (TMEV)-induced demyelination and neurological disease, and that CD4+ T cells make a greater contribution to IFN-γ-mediated protection. To determine the cellular targets of IFN-γ-mediated responses, we used adoptive transfer studies and bone marrow chimerism to generate mice in which either hematopoietic or somatic cells lacked the ability to express IFN-γ receptor. We demonstrate that IFN-γ receptor must be present on central nervous system glia, but not bone marrow-derived lymphocytes, in order to maintain resistance to TMEV-induced demyelination. PMID:11857334

Stereophysicochemical variability plots highlight conserved antigenic areas in Flaviviruses

PubMed Central

Schein, Catherine H; Zhou, Bin; Braun, Werner

2005-01-01

Background Flaviviruses, which include Dengue (DV) and West Nile (WN), mutate in response to immune system pressure. Identifying escape mutants, variant progeny that replicate in the presence of neutralizing antibodies, is a common way to identify functionally important residues of viral proteins. However, the mutations typically occur at variable positions on the viral surface that are not essential for viral replication. Methods are needed to determine the true targets of the neutralizing antibodies. Results Stereophysicochemical variability plots (SVPs), 3-D images of protein structures colored according to variability, as determined by our PCPMer program, were used to visualize residues conserved in their physical chemical properties (PCPs) near escape mutant positions. The analysis showed 1) that escape mutations in the flavivirus envelope protein are variable residues by our criteria and 2) two escape mutants found at the same position in many flaviviruses sit above clusters of conserved residues from different regions of the linear sequence. Conservation patterns in T-cell epitopes in the NS3- protease suggest a similar mechanism of immune system evasion. Conclusion The SVPs add another dimension to structurally defining the binding sites of neutralizing antibodies. They provide a useful aid for determining antigenically important regions and designing vaccines. PMID:15845145

Molecular basis of viral and microbial pathogenesis

DOE Office of Scientific and Technical Information (OSTI.GOV)

Rott, R.; Goebel, W.

1988-01-01

The contents of this book are: Correlation Between Viroid Structure and Pathogenicty; Antigenicity of the Influenza Haemagglutinia Membrane Glycoprotein; Viral Glycoproteins as Determinants of Pathogenicity; Virus Genes Involved in Host Range and Pathogenicity; Molecular Heterogenetiy of Pathogenic Herpus Viruses; Recombination of Foreign (Viral) DNA with Host Genome: Studies in Vivo and in a Cell-Free system; Disorders of Cellular Neuro-Functions by Persistent Viral Infection; Pathogenic Aspects of Measles Virus-Persistent Infections in Man; Analysis of the Dual Lineage Specificity of E26 Avian Leukemia Virus; Mx Gene Control of Influenza Virus Susceptibility; Shiga and Shika-Like Toxins: A Family of Related Cytokinons; and Molecularmore » Mechanisms of Pathogenicity in Shigella Flexneri.« less

Detection of intracellular canine distemper virus antigen in mink inoculated with an attenuated or a virulent strain of canine distemper virus.

PubMed

Blixenkrone-Møller, M

1989-09-01

Using an indirect immunofluorescence technique, the distribution of viral antigen in various tissues and blood mononuclear leukocytes was studied in wild mink, either vaccinated with an attenuated vaccine strain of canine distemper virus (CDV) or experimentally inoculated with the virulent Snyder-Hill strain of CDV. Viral antigen was detected in cells of the lymphoid system 6 to 12 days after vaccination. From 2 to 3 days after inoculation with the virulent strain, CDV antigen was demonstrated in cells of the lymphoid system and, during the incubation period, the antigen had spread to the epithelia and brain at days 6 and 12, respectively. In clinical cases of acute fatal canine distemper, the viral antigen was detected in a wide variety of tissues, including the cells of the lymphoid system, epithelial cells of skin, mucous membranes, lung, kidney, and cells of the CNS. The diagnostic importance of CDV antigen detection is discussed on the basis of these findings.

Mutational analysis of polyomavirus small-T-antigen functions in productive infection and in transformation.

PubMed Central

Martens, I; Nilsson, S A; Linder, S; Magnusson, G

1989-01-01

The function of polyomavirus small T antigen in productive infection and in transformation was studied. Transfection of permissive mouse cells with mixtures of mutants that express only one type of T antigen showed that small T antigen increased large-T-antigen-dependent viral DNA synthesis approximately 10-fold. Under the same conditions, small T antigen was also essential for the formation of infectious virus particles. To analyze these activities of small T antigen, mutants producing protein with single amino acid replacements were constructed. Two mutants, bc1073 and bc1075, were characterized. Although both mutations led to the substitution of amino acid residues of more than one T antigen, the phenotype of both mutants was associated with alterations of the small T antigen. Both mutant proteins had lost their activity in the maturation of infectious virus particles. The bc1075 but not the bc1073 small T antigen had also lost its ability to stimulate viral DNA synthesis in mouse 3T6 cells. Finally, both mutants retained a third activity of small T antigen: to confer on rat cells also expressing middle T antigen the ability to grow efficiently in semisolid medium. The phenotypes of the mutants in these three assays suggest that small T antigen has at least three separate functions. Images PMID:2704075

Human Parainfluenza Virus-3 can be Targeted by Rapidly ex vivo Expanded T-Lymphocytes

PubMed Central

McLaughlin, Lauren P.; Lang, Haili; Williams, Elizabeth; Wright, Kaylor E.; Powell, Allison; Cruz, Conrad R; Colberg-Poley, Anamaris M.; Barese, Cecilia; Hanley, Patrick J.; Bollard, Catherine M.; Keller, Michael D.

2016-01-01

Background Human Parainfluenza virus-3 (HPIV) is a common cause of respiratory infection in immunocompromised patients, and presently has no effective therapies. Virus-specific T-cell therapy has been successful for the treatment or prevention of viral infections in immunocompromised patients, but requires determination of T-cell antigens on targeted viruses. Methods HPIV3-specific T cells were expanded from peripheral blood of healthy donors using a rapid generation protocol targeting four HPIV3 proteins. Immunophenotyping was performed by flow cytometry. Viral specificity was determined by IFN-γ ELISpot, intracellular cytokine staining, and cytokine measurements from culture supernatants by Luminex assay. Cytotoxic activity was tested by 51Cr release and CD107a mobilization assays. Virus-specific T-cells targeting 6 viruses were then produced by rapid protocol, and the phenotype of HPIV3-specific T-cells was determined by immunomagnetic sorting for IFN-γ producing cells. Results HPIV3-specific T cells were expanded from 13 healthy donors. HPIV3-specific T-cells showed a CD4+ predominance (mean CD4:CD8 ratio 2.89), and demonstrated specificity for multiple HPIV3 antigens. The expanded T-cells were polyfunctional based on cytokine production, but only had a minor cytotoxic component. T cells targeting six viruses in a single product similarly showed HPIV3 specificity, with a predominant effector memory phenotype (CD3+/CD45RA-/CCR7-) in responder cells. Discussion HPIV3-specific T cells can be produced using a rapid ex vivo protocol from healthy donors and are predominantly CD4+ T-cells with Th1 activity. HPIV3 epitopes can also be successfully targeted alongside multiple other viral epitopes in production of 6-virus T-cells, without loss of HPIV3 specificity. These products may be clinically beneficial to combat HPIV3 infections by adoptive T-cell therapy in immune compromised patients. PMID:27692559

Oral Delivery of Probiotics Expressing Dendritic Cell-Targeting Peptide Fused with Porcine Epidemic Diarrhea Virus COE Antigen: A Promising Vaccine Strategy against PEDV.

PubMed

Wang, Xiaona; Wang, Li; Huang, Xuewei; Ma, Sunting; Yu, Meiling; Shi, Wen; Qiao, Xinyuan; Tang, Lijie; Xu, Yigang; Li, Yijing

2017-10-25

Porcine epidemic diarrhea virus (PEDV), an enteric coronavirus, is the causative agent of porcine epidemic diarrhea (PED) that damages intestinal epithelial cells and results in severe diarrhea and dehydration in neonatal suckling pigs with up to 100% mortality. The oral vaccine route is reported as a promising approach for inducing protective immunity against PEDV invasion. Furthermore, dendritic cells (DCs), professional antigen-presenting cells, link humoral and cellular immune responses for homeostasis of the intestinal immune environment. In this study, in order to explore an efficient oral vaccine against PEDV infection, a mucosal DC-targeting oral vaccine was developed using Lactobacillus casei to deliver the DC-targeting peptide (DCpep) fused with the PEDV core neutralizing epitope (COE) antigen. This probiotic vaccine could efficiently elicit secretory immunoglobulin A (SIgA)-based mucosal and immunoglobulin G (IgG)-based humoral immune responses via oral vaccination in vivo. Significant differences ( p < 0.05) in the immune response levels were observed between probiotics expressing the COE-DCpep fusion protein and COE antigen alone, suggesting better immune efficiency of the probiotics vaccine expressing the DC-targeting peptide fused with PEDV COE antigen. This mucosal DC-targeting oral vaccine delivery effectively enhances vaccine antigen delivery efficiency, providing a useful strategy to induce efficient immune responses against PEDV infection.

Mannose-pepstatin conjugates as targeted inhibitors of antigen processing.

PubMed

Free, Paul; Hurley, Christopher A; Kageyama, Takashi; Chain, Benjamin M; Tabor, Alethea B

2006-05-07

The molecular details of antigen processing, including the identity of the enzymes involved, their intracellular location and their substrate specificity, are still incompletely understood. Selective inhibition of proteolytic antigen processing enzymes such as cathepsins D and E, using small molecular inhibitors such as pepstatin, has proven to be a valuable tool in investigating these pathways. However, pepstatin is poorly soluble in water and has limited access to the antigen processing compartment in antigen presenting cells. We have synthesised mannose-pepstatin conjugates, and neomannosylated BSA-pepstatin conjugates, as tools for the in vivo study of the antigen processing pathway. Conjugation to mannose and to neomannosylated BSA substantially improved the solubility of the conjugates relative to pepstatin. The mannose-pepstatin conjugates showed no reduction in inhibition of cathepsin E, whereas the neomannosylated BSA-pepstatin conjugates showed some loss of inhibition, probably due to steric factors. However, a neomannosylated BSA-pepstatin conjugate incorporating a cleavable disulfide linkage between the pepstatin and the BSA showed the best uptake to dendritic cells and the best inhibition of antigen processing.

A single point in protein trafficking by Plasmodium falciparum determines the expression of major antigens on the surface of infected erythrocytes targeted by human antibodies.

PubMed

Chan, Jo-Anne; Howell, Katherine B; Langer, Christine; Maier, Alexander G; Hasang, Wina; Rogerson, Stephen J; Petter, Michaela; Chesson, Joanne; Stanisic, Danielle I; Duffy, Michael F; Cooke, Brian M; Siba, Peter M; Mueller, Ivo; Bull, Peter C; Marsh, Kevin; Fowkes, Freya J I; Beeson, James G

2016-11-01

Antibodies to blood-stage antigens of Plasmodium falciparum play a pivotal role in human immunity to malaria. During parasite development, multiple proteins are trafficked from the intracellular parasite to the surface of P. falciparum-infected erythrocytes (IEs). However, the relative importance of different proteins as targets of acquired antibodies, and key pathways involved in trafficking major antigens remain to be clearly defined. We quantified antibodies to surface antigens among children, adults, and pregnant women from different malaria-exposed regions. We quantified the importance of antigens as antibody targets using genetically engineered P. falciparum with modified surface antigen expression. Genetic deletion of the trafficking protein skeleton-binding protein-1 (SBP1), which is involved in trafficking the surface antigen PfEMP1, led to a dramatic reduction in antibody recognition of IEs and the ability of human antibodies to promote opsonic phagocytosis of IEs, a key mechanism of parasite clearance. The great majority of antibody epitopes on the IE surface were SBP1-dependent. This was demonstrated using parasite isolates with different genetic or phenotypic backgrounds, and among antibodies from children, adults, and pregnant women in different populations. Comparisons of antibody reactivity to parasite isolates with SBP1 deletion or inhibited PfEMP1 expression suggest that PfEMP1 is the dominant target of acquired human antibodies, and that other P. falciparum IE surface proteins are minor targets. These results establish SBP1 as part of a critical pathway for the trafficking of major surface antigens targeted by human immunity, and have key implications for vaccine development, and quantifying immunity in populations.

Targeting HIV-1 gp41-induced fusion and pathogenesis for anti-viral therapy.

PubMed

Garg, Himanshu; Viard, Mathias; Jacobs, Amy; Blumenthal, Robert

2011-12-01

HIV gp41 is a metastable protein whose native conformation is maintained in the form of a heterodimer with gp120. The non-covalently associated gp41/gp120 complex forms a trimer on the virus surface. As gp120 engages with HIV's receptor, CD4, and coreceptor, CXCR4 or CCR5, gp41 undergoes several conformational changes resulting in fusion between the viral and cellular membranes. Several lipophilic and amphiphilic domains have been shown to be critical in that process. While the obvious function of gp41 in viral entry is well-established its role in cellular membrane fusion and the link with pathogenesis are only now beginning to appear. Recent targeting of gp41 via fusion inhibitors has revealed an important role of this protein not only in viral entry but also in bystander apoptosis and HIV pathogenesis. Studies by our group and others have shown that the phenomenon of gp41-mediated hemifusion initiates apoptosis in bystander cells and correlates with virus pathogenesis. More interestingly, recent clinical evidence suggests that gp41 mutants arising after Enfuvirtide therapy are associated with CD4 cell increase and immunological benefits. This has in turn been correlated to a decrease in bystander apoptosis in our in vitro as well as in vivo assays. Although a great deal of work has been done to unravel HIV-1 gp41-mediated fusion mechanisms, the factors that regulate gp41-mediated fusion versus hemifusion and the mechanism by which hemifusion initiates bystander apoptosis are not fully understood. Further insight into these issues will open new avenues for drug development making gp41 a critical anti-HIV target both for neutralization and virus attenuation.

Environmental Factors Contribute to β Cell Endoplasmic Reticulum Stress and Neo-Antigen Formation in Type 1 Diabetes

PubMed Central

Marré, Meghan L.; Piganelli, Jon D.

2017-01-01

Type 1 diabetes (T1D) is an autoimmune disease in which immune-mediated targeting and destruction of insulin-producing pancreatic islet β cells leads to chronic hyperglycemia. There are many β cell proteins that are targeted by autoreactive T cells in their native state. However, recent studies have demonstrated that many β cell proteins are recognized as neo-antigens following posttranslational modification (PTM). Although modified neo-antigens are well-established targets of pathology in other autoimmune diseases, the effects of neo-antigens in T1D progression and the mechanisms by which they are generated are not well understood. We have demonstrated that PTM occurs during endoplasmic reticulum (ER) stress, a process to which β cells are uniquely susceptible due to the high rate of insulin production in response to dynamic glucose sensing. In the context of genetic susceptibility to autoimmunity, presentation of these modified neo-antigens may activate autoreactive T cells and cause pathology. However, inherent β cell ER stress and protein PTM do not cause T1D in every genetically susceptible individual, suggesting the contribution of additional factors. Indeed, many environmental factors, such as viral infection, chemicals, or inflammatory cytokines, are associated with T1D onset, but the mechanisms by which these factors lead to disease onset remain unknown. Since these environmental factors also cause ER stress, exposure to these factors may enhance production of neo-antigens, therefore boosting β cell recognition by autoreactive T cells and exacerbating T1D pathogenesis. Therefore, the combined effects of physiological ER stress and the stress that is induced by environmental factors may lead to breaks in peripheral tolerance, contribute to antigen spread, and hasten disease onset. This Hypothesis and Theory article summarizes what is currently known about ER stress and protein PTM in autoimmune diseases including T1D and proposes a role for

Systemic surfaceome profiling identifies target antigens for immune-based therapy in subtypes of advanced prostate cancer

PubMed Central

Lee, John K.; Bangayan, Nathanael J.; Chai, Timothy; Smith, Bryan A.; Pariva, Tiffany E.; Yun, Sangwon; Vashisht, Ajay; Zhang, Qingfu; Park, Jung Wook; Corey, Eva; Huang, Jiaoti; Wohlschlegel, James; Witte, Owen N.

2018-01-01

Prostate cancer is a heterogeneous disease composed of divergent molecular and histologic subtypes, including prostate adenocarcinoma (PrAd) and neuroendocrine prostate cancer (NEPC). While PrAd is the major histology in prostate cancer, NEPC can evolve from PrAd as a mechanism of treatment resistance that involves a transition from an epithelial to a neurosecretory cancer phenotype. Cell surface markers are often associated with specific cell lineages and differentiation states in normal development and cancer. Here, we show that PrAd and NEPC can be broadly discriminated by cell-surface profiles based on the analysis of prostate cancer gene expression datasets. To overcome a dependence on predictions of human cell-surface genes and an assumed correlation between mRNA levels and protein expression, we integrated transcriptomic and cell-surface proteomic data generated from a panel of prostate cancer cell lines to nominate cell-surface markers associated with these cancer subtypes. FXYD3 and CEACAM5 were validated as cell-surface antigens enriched in PrAd and NEPC, respectively. Given the lack of effective treatments for NEPC, CEACAM5 appeared to be a promising target for cell-based immunotherapy. As a proof of concept, engineered chimeric antigen receptor T cells targeting CEACAM5 induced antigen-specific cytotoxicity in NEPC cell lines. Our findings demonstrate that the surfaceomes of PrAd and NEPC reflect unique cancer differentiation states and broadly represent vulnerabilities amenable to therapeutic targeting. PMID:29686080

The influence of TAP1 and TAP2 gene polymorphisms on TAP function and its inhibition by viral immune evasion proteins.

PubMed

Praest, P; Luteijn, R D; Brak-Boer, I G J; Lanfermeijer, J; Hoelen, H; Ijgosse, L; Costa, A I; Gorham, R D; Lebbink, R J; Wiertz, E J H J

2018-06-04

Herpesviruses encode numerous immune evasion molecules that interfere with the immune system, particularly with certain stages in the MHC class I antigen presentation pathway. In this pathway, the transporter associated with antigen processing (TAP) is a frequent target of viral immune evasion strategies. This ER-resident transporter is composed of the proteins TAP1 and TAP2, and plays a crucial role in the loading of viral peptides onto MHC class I molecules. Several variants of TAP1 and TAP2 occur in the human population, some of which are linked to autoimmune disorders and susceptibility to infections. Here, we assessed the influence of naturally occurring TAP variants on peptide transport and MHC class I expression. In addition, we tested the inhibitory capacity of three viral immune evasion proteins, the TAP inhibitors US6 from human cytomegalovirus, ICP47 from herpes simplex virus type 1 and BNLF2a from Epstein-Barr virus, for a series of TAP1 and TAP2 variants. Our results suggest that these TAP polymorphisms have no or limited effect on peptide transport or MHC class I expression. Furthermore, our study indicates that the herpesvirus-encoded TAP inhibitors target a broad spectrum of TAP variants; inhibition of TAP is not affected by the naturally occurring polymorphisms of TAP tested in this study. Our findings suggest that the long-term coevolution of herpesviruses and their host did not result in selection of inhibitor-resistant TAP variants in the human population. Copyright © 2018. Published by Elsevier Ltd.

Targeting of viral interleukin-10 with an antibody fragment specific to damaged arthritic cartilage improves its therapeutic potency

PubMed Central

2014-01-01

Introduction We previously demonstrated that a single-chain fragment variable (scFv) specific to collagen type II (CII) posttranslationally modified by reactive oxygen species (ROS) can be used to target anti-inflammatory therapeutics specifically to inflamed arthritic joints. The objective of the present study was to demonstrate the superior efficacy of anti-inflammatory cytokines when targeted to inflamed arthritic joints by the anti-ROS modified CII (anti-ROS-CII) scFv in a mouse model of arthritis. Methods Viral interleukin-10 (vIL-10) was fused to anti-ROS-CII scFv (1-11E) with a matrix-metalloproteinase (MMP) cleavable linker to create 1-11E/vIL-10 fusion. Binding of 1-11E/vIL-10 to ROS-CII was determined by enzyme-linked immunosorbent assay (ELISA), Western blotting, and immune-staining of arthritic cartilage, whereas vIL-10 bioactivity was evaluated in vitro by using an MC-9 cell-proliferation assay. Specific in vivo localization and therapeutic efficacy of 1-11E/vIL-10 was tested in the mouse model of antigen-induced arthritis. Results 1-11E/vIL-10 bound specifically to ROS-CII and to damaged arthritic cartilage. Interestingly, the in vitro vIL-10 activity in the fusion protein was observed only after cleavage with MMP-1. When systemically administered to arthritic mice, 1-11E/vIL-10 localized specifically to the arthritic knee, with peak accumulation observed after 3 days. Moreover, 1-11E/vIL-10 reduced inflammation significantly quicker than vIL-10 fused to the control anti-hen egg lysozyme scFv (C7/vIL10). Conclusions Targeted delivery of anti-inflammatory cytokines potentiates their anti-arthritic action in a mouse model of arthritis. Our results further support the hypothesis that targeting biotherapeutics to arthritic joints may be extended to include anti-inflammatory cytokines that lack efficacy when administered systemically. PMID:25029910

Skin Dendritic Cell Targeting via Microneedle Arrays Laden with Antigen-Encapsulated Poly-d,l-lactide-co-Glycolide Nanoparticles Induces Efficient Antitumor and Antiviral Immune Responses

PubMed Central

2013-01-01

The efficacious delivery of antigens to antigen-presenting cells (APCs), in particular, to dendritic cells (DCs), and their subsequent activation remains a significant challenge in the development of effective vaccines. This study highlights the potential of dissolving microneedle (MN) arrays laden with nanoencapsulated antigen to increase vaccine immunogenicity by targeting antigen specifically to contiguous DC networks within the skin. Following in situ uptake, skin-resident DCs were able to deliver antigen-encapsulated poly-d,l-lactide-co-glycolide (PGLA) nanoparticles to cutaneous draining lymph nodes where they subsequently induced significant expansion of antigen-specific T cells. Moreover, we show that antigen-encapsulated nanoparticle vaccination via microneedles generated robust antigen-specific cellular immune responses in mice. This approach provided complete protection in vivo against both the development of antigen-expressing B16 melanoma tumors and a murine model of para-influenza, through the activation of antigen-specific cytotoxic CD8+ T cells that resulted in efficient clearance of tumors and virus, respectively. In addition, we show promising findings that nanoencapsulation facilitates antigen retention into skin layers and provides antigen stability in microneedles. Therefore, the use of biodegradable polymeric nanoparticles for selective targeting of antigen to skin DC subsets through dissolvable MNs provides a promising technology for improved vaccination efficacy, compliance, and coverage. PMID:23373658

High-efficiency targeted editing of large viral genomes by RNA-guided nucleases.

PubMed

Bi, Yanwei; Sun, Le; Gao, Dandan; Ding, Chen; Li, Zhihua; Li, Yadong; Cun, Wei; Li, Qihan

2014-05-01

A facile and efficient method for the precise editing of large viral genomes is required for the selection of attenuated vaccine strains and the construction of gene therapy vectors. The type II prokaryotic CRISPR-Cas (clustered regularly interspaced short palindromic repeats (CRISPR)-associated (Cas)) RNA-guided nuclease system can be introduced into host cells during viral replication. The CRISPR-Cas9 system robustly stimulates targeted double-stranded breaks in the genomes of DNA viruses, where the non-homologous end joining (NHEJ) and homology-directed repair (HDR) pathways can be exploited to introduce site-specific indels or insert heterologous genes with high frequency. Furthermore, CRISPR-Cas9 can specifically inhibit the replication of the original virus, thereby significantly increasing the abundance of the recombinant virus among progeny virus. As a result, purified recombinant virus can be obtained with only a single round of selection. In this study, we used recombinant adenovirus and type I herpes simplex virus as examples to demonstrate that the CRISPR-Cas9 system is a valuable tool for editing the genomes of large DNA viruses.

High-Efficiency Targeted Editing of Large Viral Genomes by RNA-Guided Nucleases

PubMed Central

Gao, Dandan; Ding, Chen; Li, Zhihua; Li, Yadong; Cun, Wei; Li, Qihan

2014-01-01

A facile and efficient method for the precise editing of large viral genomes is required for the selection of attenuated vaccine strains and the construction of gene therapy vectors. The type II prokaryotic CRISPR-Cas (clustered regularly interspaced short palindromic repeats (CRISPR)-associated (Cas)) RNA-guided nuclease system can be introduced into host cells during viral replication. The CRISPR-Cas9 system robustly stimulates targeted double-stranded breaks in the genomes of DNA viruses, where the non-homologous end joining (NHEJ) and homology-directed repair (HDR) pathways can be exploited to introduce site-specific indels or insert heterologous genes with high frequency. Furthermore, CRISPR-Cas9 can specifically inhibit the replication of the original virus, thereby significantly increasing the abundance of the recombinant virus among progeny virus. As a result, purified recombinant virus can be obtained with only a single round of selection. In this study, we used recombinant adenovirus and type I herpes simplex virus as examples to demonstrate that the CRISPR-Cas9 system is a valuable tool for editing the genomes of large DNA viruses. PMID:24788700

The influence of antigen targeting to sub-cellular compartments on the anti-allergic potential of a DNA vaccine.

PubMed

Weinberger, Esther E; Isakovic, Almedina; Scheiblhofer, Sandra; Ramsauer, Christina; Reiter, Katrin; Hauser-Kronberger, Cornelia; Thalhamer, Josef; Weiss, Richard

2013-12-09

Gene vaccines offer attractive rationales for prophylactic as well as therapeutic treatments of type I allergies. DNA and mRNA vaccines have been shown to prevent from allergic sensitization and to counterbalance established allergic immune reactions. Recent advances in gene vaccine manipulation offer additional opportunities for modulation of T helper cell profiles by specific targeting of cellular compartments. DNA vaccines encoding the major birch pollen allergen Bet v 1.0101 were equipped with different leader sequences to shuttle the antigen to lysosomes (LIMP-II), to trigger cellular secretion (hTPA), or to induce proteasomal degradation via forced ubiquitination (ubi). Mice were pre-vaccinated with these constructs and the protective efficacy was tested by subcutaneous Th2-promoting challenges, followed by allergen inhalation. IgG antibody subclass distribution and allergen-specific IgE as well as cytokine profiles from re-stimulated splenocytes and from BALFs were assessed. The cellular composition of BALFs, and lung resistance and compliance were determined. Immunization with all targeting variants protected from allergic sensitization, i.e. IgE induction, airway hyperresponsiveness, lung inflammation, and systemic and local Th2 cytokine expression. Surprisingly, protection did not clearly correlate with the induction of a systemic Th1 cytokine profile, but rather with proliferating CD4+ CD25+ FoxP3+ T regulatory cells in splenocyte cultures. Targeting the allergen to proteasomal or lysosomal degradation severely down-regulated antibody induction after vaccination, while T cell responses remained unaffected. Although secretion of antigen promoted the highest numbers of Th1 cells, this vaccine type was the least efficient in suppressing the establishment of an allergic immune response. This comparative analysis highlights the modulatory effect of antigen targeting on the resulting immune response, with a special emphasis on prophylactic anti-allergy DNA

Simultaneous targeting of tumor antigens and the tumor vasculature using T lymphocyte transfer synergize to induce regression of established tumors in mice.

PubMed

Chinnasamy, Dhanalakshmi; Tran, Eric; Yu, Zhiya; Morgan, Richard A; Restifo, Nicholas P; Rosenberg, Steven A

2013-06-01

Most systemic cancer therapies target tumor cells directly, although there is increasing interest in targeting the tumor stroma that can comprise a substantial portion of the tumor mass. We report here a synergy between two T-cell therapies, one directed against the stromal tumor vasculature and the other directed against antigens expressed on the tumor cell. Simultaneous transfer of genetically engineered syngeneic T cells expressing a chimeric antigen receptor targeting the VEGF receptor-2 (VEGFR2; KDR) that is overexpressed on tumor vasculature and T-cells specific for the tumor antigens gp100 (PMEL), TRP-1 (TYRP1), or TRP-2 (DCT) synergistically eradicated established B16 melanoma tumors in mice and dramatically increased the tumor-free survival of mice compared with treatment with either cell type alone or T cells coexpressing these two targeting molecules. Host lymphodepletion before cell transfer was required to mediate the antitumor effect. The synergistic antitumor response was accompanied by a significant increase in the infiltration and expansion and/or persistence of the adoptively transferred tumor antigen-specific T cells in the tumor microenvironment and thus enhanced their antitumor potency. The data presented here emphasize the possible beneficial effects of combining antiangiogenic with tumor-specific immunotherapeutic approaches for the treatment of patients with cancer. ©2013 AACR.

Mutational Inactivation of Herpes Simplex Virus 1 MicroRNAs Identifies Viral mRNA Targets and Reveals Phenotypic Effects in Culture

PubMed Central

Flores, Omar; Nakayama, Sanae; Whisnant, Adam W.; Javanbakht, Hassan; Cullen, Bryan R.

2013-01-01

Herpes simplex virus 1 (HSV-1), a ubiquitous human pathogen, expresses several viral microRNAs (miRNAs). These, along with the latency-associated transcript, represent the only viral RNAs detectable in latently infected neuronal cells. Here, for the first time, we analyze which HSV-1 miRNAs are loaded into the RNA-induced silencing complex (RISC), the key effector of miRNA function. Only 9 of the 17 reported HSV-1 miRNAs, i.e., miR-H1 to miR-H8 plus miR-H11, were found to actually load into the RISC. Surprisingly, this analysis also revealed that HSV-1 miRNAs loaded into the RISC with efficiencies that differed widely; <1% of the miR-H1-3p miRNA detectable in HSV-1-infected cells was loaded into the RISC. Analysis of HSV-1 mutants individually lacking the viral miR-H2, miR-H3, or miR-H4 miRNA revealed that loss of these miRNAs affected the rate of replication of HSV-1 in neuronal cells but not in fibroblasts. Analysis of mRNA and protein expression, as well as assays mapping viral miRNA binding sites in infected cells, showed that endogenous HSV-1 miR-H2 binds to viral ICP0 mRNA and inhibits its expression, while endogenous miR-H4 inhibits the expression of the viral ICP34.5 gene. In contrast, no viral mRNA target for miR-H3 could be detected, even though miR-H3, like miR-H4, is perfectly complementary to ICP34.5 mRNA. Together, these data demonstrate that endogenous HSV-1 miRNA expression can significantly alter viral replication in culture, and they also identify two viral mRNA targets for miR-H2 and miR-H4 that can partially explain this phenotype. PMID:23536669

Development and Evaluation of Single Domain Antibodies for Vaccinia and the L1 Antigen

PubMed Central

Walper, Scott A.; Liu, Jinny L.; Zabetakis, Daniel; Anderson, George P.; Goldman, Ellen R.

2014-01-01

There is ongoing interest to develop high affinity, thermal stable recognition elements to replace conventional antibodies in biothreat detection assays. As part of this effort, single domain antibodies that target vaccinia virus were developed. Two llamas were immunized with killed viral particles followed by boosts with the recombinant membrane protein, L1, to stimulate the immune response for envelope and membrane proteins of the virus. The variable domains of the induced heavy chain antibodies were selected from M13 phage display libraries developed from isolated RNA. Selection via biopanning on the L1 antigen produced single domain antibodies that were specific and had affinities ranging from 4×10−9 M to 7.0×10−10 M, as determined by surface plasmon resonance. Several showed good ability to refold after heat denaturation. These L1-binding single domain antibodies, however, failed to recognize the killed vaccinia antigen. Useful vaccinia binding single domain antibodies were isolated by a second selection using the killed virus as the target. The virus binding single domain antibodies were incorporated in sandwich assays as both capture and tracer using the MAGPIX system yielding limits of detection down to 4×105 pfu/ml, a four-fold improvement over the limit obtained using conventional antibodies. This work demonstrates the development of anti-vaccinia single domain antibodies and their incorporation into sandwich assays for viral detection. It also highlights the properties of high affinity and thermal stability that are hallmarks of single domain antibodies. PMID:25211488

Regulatory T Cells in Autoimmune and Viral Chronic Hepatitis

PubMed Central

Lapierre, Pascal; Lamarre, Alain

2015-01-01

In both autoimmune liver disease and chronic viral hepatitis, the injury results from an immune-mediated cytotoxic T cell response to liver cells. As such, it is not surprising that CD4+ regulatory T cells, a key regulatory population of T cells able to curb immune responses, could be involved in both autoimmune hepatitis and chronic viral hepatitis. The liver can induce the conversion of naïve CD4+ T cells to CD4+ regulatory T cells and induce tolerance to locally expressed antigens. This tolerance mechanism is carefully regulated in physiological conditions but any imbalance could be pathological. An overly tolerant immune response can lead to chronic infections while an overreactive and unbridled immune response can lead to autoimmune hepatitis. With the recent advent of monoclonal antibodies able to target regulatory T cells (daclizumab) and improve immune responses and several ongoing clinical trials analysing the impact of regulatory T cell infusion on autoimmune liver disease or liver transplant tolerance, modulation of immunological tolerance through CD4+ regulatory T cells could be a key element of future immunotherapies for several liver diseases allowing restoring the balance between proper immune responses and tolerance.   PMID:26106627

Glioma antigen.

PubMed

Toda, Masahiro

2012-01-01

Because several antigenic peptides of human tumors that are recognized by T-lymphocytes have been identified, immune responses against cancer can now be artificially manipulated. Furthermore, since T-lymphocytes have been found to play an important role in the rejection of tumors by the host and also to have antigen-specific proliferative potentials and memory mechanisms, T-lymphocytes are thought to play a central role in cancer vaccination. Although multidisciplinary therapies have been attempted for the treatment of gliomas, the results remain unsatisfactory. For the development of new therapies against gliomas, it is required to identify tumor antigens as targets for specific immunotherapy. In this chapter, recent progress in research on glioma antigens is described.

Intracellular chromobody delivery by mesoporous silica nanoparticles for antigen targeting and visualization in real time

PubMed Central

Chiu, Hsin-Yi; Deng, Wen; Engelke, Hanna; Helma, Jonas; Leonhardt, Heinrich; Bein, Thomas

2016-01-01

Chromobodies have recently drawn great attention as bioimaging nanotools. They offer high antigen binding specificity and affinity comparable to conventional antibodies, but much smaller size and higher stability. Chromobodies can be used in live cell imaging for specific spatio-temporal visualization of cellular processes. To date, functional application of chromobodies requires lengthy genetic manipulation of the target cell. Here, we develop multifunctional large-pore mesoporous silica nanoparticles (MSNs) as nanocarriers to directly transport chromobodies into living cells for antigen-visualization in real time. The multifunctional large-pore MSNs feature high loading capacity for chromobodies, and are efficiently taken up by cells. By functionalizing the internal MSN surface with nitrilotriacetic acid-metal ion complexes, we can control the release of His6-tagged chromobodies from MSNs in acidified endosomes and observe successful chromobody-antigen binding in the cytosol. Hence, by combining the two nanotools, chromobodies and MSNs, we establish a new powerful approach for chromobody applications in living cells. PMID:27173765

A Viral Receptor Complementation Strategy to Overcome CAV-2 Tropism for Efficient Retrograde Targeting of Neurons.

PubMed

Li, Shu-Jing; Vaughan, Alexander; Sturgill, James Fitzhugh; Kepecs, Adam

2018-06-06

Retrogradely transported neurotropic viruses enable genetic access to neurons based on their long-range projections and have become indispensable tools for linking neural connectivity with function. A major limitation of viral techniques is that they rely on cell-type-specific molecules for uptake and transport. Consequently, viruses fail to infect variable subsets of neurons depending on the complement of surface receptors expressed (viral tropism). We report a receptor complementation strategy to overcome this by potentiating neurons for the infection of the virus of interest-in this case, canine adenovirus type-2 (CAV-2). We designed AAV vectors for expressing the coxsackievirus and adenovirus receptor (CAR) throughout candidate projection neurons. CAR expression greatly increased retrograde-labeling rates, which we demonstrate for several long-range projections, including some resistant to other retrograde-labeling techniques. Our results demonstrate a receptor complementation strategy to abrogate endogenous viral tropism and thereby facilitate efficient retrograde targeting for functional analysis of neural circuits. Copyright © 2018 Elsevier Inc. All rights reserved.

Adoptive T-cell therapy for hematological malignancies using T cells gene-modified to express tumor antigen-specific receptors.

PubMed

Fujiwara, Hiroshi

2014-02-01

The functional properties of the adoptive immune response mediated by effector T lymphocytes are decisively regulated by their T-cell receptors (TCRs). Transfer of genes encoding target antigen-specific receptors enables polyclonal T cells to redirect toward cancer cells and virally infected cells expressing those defined antigens. Using this technology, a large population of redirected T cells displaying uniform therapeutic properties has been produced, powerfully advancing their clinical application as "cellular drugs" for adoptive immunotherapy against cancer. Clinically, anticancer adoptive immunotherapy using these genetically engineered T cells has an impressive and proven track record. Notable examples include the dramatic benefit of chimeric antigen receptor gene-modified T cells redirected towards B-cell lineage antigen CD19 in patients with chronic lymphocytic leukemia, and the impressive outcomes in the use of TCR gene-modified T cells redirected towards NY-ESO-1, a representative cancer-testis antigen, in patients with advanced melanoma and synovial cell sarcoma. In this review, we briefly overview the current status of this treatment option in the context of hematological malignancy, and discuss a number of challenges that still pose an obstacle to the full effectiveness of this strategy.

Bat Caliciviruses and Human Noroviruses Are Antigenically Similar and Have Overlapping Histo-Blood Group Antigen Binding Profiles.

PubMed

Kocher, Jacob F; Lindesmith, Lisa C; Debbink, Kari; Beall, Anne; Mallory, Michael L; Yount, Boyd L; Graham, Rachel L; Huynh, Jeremy; Gates, J Edward; Donaldson, Eric F; Baric, Ralph S

2018-05-22

Emerging zoonotic viral diseases remain a challenge to global public health. Recent surveillance studies have implicated bats as potential reservoirs for a number of viral pathogens, including coronaviruses and Ebola viruses. Caliciviridae represent a major viral family contributing to emerging diseases in both human and animal populations and have been recently identified in bats. In this study, we blended metagenomics, phylogenetics, homology modeling, and in vitro assays to characterize two novel bat calicivirus (BtCalV) capsid sequences, corresponding to strain BtCalV/A10/USA/2009, identified in Perimyotis subflavus near Little Orleans, MD, and bat norovirus. We observed that bat norovirus formed virus-like particles and had epitopes and receptor-binding patterns similar to those of human noroviruses. To determine whether these observations stretch across multiple bat caliciviruses, we characterized a novel bat calicivirus, BtCalV/A10/USA/2009. Phylogenetic analysis revealed that BtCalV/A10/USA/2009 likely represents a novel Caliciviridae genus and is most closely related to "recoviruses." Homology modeling revealed that the capsid sequences of BtCalV/A10/USA/2009 and bat norovirus resembled human norovirus capsid sequences and retained host ligand binding within the receptor-binding domains similar to that seen with human noroviruses. Both caliciviruses bound histo-blood group antigens in patterns that overlapped those seen with human and animal noroviruses. Taken together, our results indicate the potential for bat caliciviruses to bind histo-blood group antigens and overcome a significant barrier to cross-species transmission. Additionally, we have shown that bat norovirus maintains antigenic epitopes similar to those seen with human noroviruses, providing further evidence of evolutionary descent. Our results reiterate the importance of surveillance of wild-animal populations, especially of bats, for novel viral pathogens. IMPORTANCE Caliciviruses are

Virtual screening of the inhibitors targeting at the viral protein 40 of Ebola virus.

PubMed

Karthick, V; Nagasundaram, N; Doss, C George Priya; Chakraborty, Chiranjib; Siva, R; Lu, Aiping; Zhang, Ge; Zhu, Hailong

2016-02-17

The Ebola virus is highly pathogenic and destructive to humans and other primates. The Ebola virus encodes viral protein 40 (VP40), which is highly expressed and regulates the assembly and release of viral particles in the host cell. Because VP40 plays a prominent role in the life cycle of the Ebola virus, it is considered as a key target for antiviral treatment. However, there is currently no FDA-approved drug for treating Ebola virus infection, resulting in an urgent need to develop effective antiviral inhibitors that display good safety profiles in a short duration. This study aimed to screen the effective lead candidate against Ebola infection. First, the lead molecules were filtered based on the docking score. Second, Lipinski rule of five and the other drug likeliness properties are predicted to assess the safety profile of the lead candidates. Finally, molecular dynamics simulations was performed to validate the lead compound. Our results revealed that emodin-8-beta-D-glucoside from the Traditional Chinese Medicine Database (TCMD) represents an active lead candidate that targets the Ebola virus by inhibiting the activity of VP40, and displays good pharmacokinetic properties. This report will considerably assist in the development of the competitive and robust antiviral agents against Ebola infection.

MERS-CoV and H5N1 influenza virus antagonize antigen presentation by altering the epigenetic landscape

DOE PAGES

Menachery, Vineet D.; Schafer, Alexandra; Burnum-Johnson, Kristin E.; ...

2018-01-16

Convergent evolution dictates that diverse groups of viruses will target both similar and distinct host pathways in order to manipulate the immune response and improve infection. In this study, we sought to leverage this uneven viral antagonism to identify critical host factors that govern disease outcome. Utilizing a systems based approach, we examined differential regulation of IFNγ dependent genes following infection with highly pathogenic viruses including influenza (H5N1-VN1203, H1N1-CA04) and coronaviruses (SARS-CoV, MERS-CoV). Categorizing by function, we observed down regulation of genes associated with antigen presentation following both H5N1-VN1203 and MERS-CoV infection. Further examination revealed global down regulation of antigenmore » presentation genes and was confirmed by proteomics for both H5N1-VN1203 and MERS-CoV infection. Importantly, epigenetic analysis suggested that DNA methylation rather than histone modification plays a crucial role in MERS-CoV mediated antagonism of antigen presentation genes; in contrast, H5N1-VN1203 likely utilizes a combination of epigenetic mechanisms to target antigen presentation. Altogether, the results indicate a common approach utilized by H5N1-VN1203 and MERS-CoV to modulate antigen presentation and the host adaptive immune response.« less

MERS-CoV and H5N1 influenza virus antagonize antigen presentation by altering the epigenetic landscape

DOE Office of Scientific and Technical Information (OSTI.GOV)

Menachery, Vineet D.; Schäfer, Alexandra; Burnum-Johnson, Kristin E.

Convergent evolution dictates that diverse groups of viruses will target both similar and distinct host pathways in order to manipulate the immune response and improve infection. In this study, we sought to leverage this uneven viral antagonism to identify critical host factors that govern disease outcome. Utilizing a systems based approach, we examined differential regulation of IFNγ dependent genes following infection with highly pathogenic viruses including influenza (H5N1-VN1203, H1N1-CA04) and coronaviruses (SARS-CoV, MERS-CoV). Categorizing by function, we observed down regulation of genes associated with antigen presentation following both H5N1-VN1203 and MERS-CoV infection. Further examination revealed global down regulation of antigenmore » presentation genes and was confirmed by proteomics for both H5N1-VN1203 and MERS-CoV infection. Importantly, epigenetic analysis suggested that DNA methylation rather than histone modification plays a crucial role in MERS-CoV mediated antagonism of antigen presentation genes; in contrast, H5N1-VN1203 likely utilizes a combination of epigenetic mechanisms to target antigen presentation. Together, the results indicate a common approach utilized by H5N1-VN1203 and MERS-CoV to modulate antigen presentation and the host adaptive immune response.« less

The Mechanism of Synchronous Precise Regulation of Two Shrimp White Spot Syndrome Virus Targets by a Viral MicroRNA

PubMed Central

He, Yaodong; Ma, Tiantian; Zhang, Xiaobo

2017-01-01

MicroRNAs (miRNAs), important factors in animal innate immunity, suppress the expressions of their target genes by binding to target mRNA’s 3′ untranslated regions (3′UTRs). However, the mechanism of synchronous regulation of multiple targets by a single miRNA remains unclear. In this study, the interaction between a white spot syndrome virus (WSSV) miRNA (WSSV-miR-N32) and its two viral targets (wsv459 and wsv322) was characterized in WSSV-infected shrimp. The outcomes indicated that WSSV-encoded miRNA (WSSV-miR-N32) significantly inhibited virus infection by simultaneously targeting wsv459 and wsv322. The silencing of wsv459 or wsv322 by siRNA led to significant decrease of WSSV copies in shrimp, showing that the two viral genes were required for WSSV infection. WSSV-miR-N32 could mediate 5′–3′ exonucleolytic digestion of its target mRNAs, which stopped at the sites of target mRNA 3′UTRs close to the sequence complementary to the miRNA seed sequence. The complementary bases (to the target mRNA sequence) of a miRNA 9th–18th non-seed sequence were essential for the miRNA targeting. Therefore, our findings presented novel insights into the mechanism of miRNA-mediated suppression of target gene expressions, which would be helpful for understanding the roles of miRNAs in innate immunity of invertebrate. PMID:29230209

Development of prostate specific membrane antigen targeted ultrasound microbubbles using bioorthogonal chemistry

PubMed Central

Zlitni, Aimen; Yin, Melissa; Janzen, Nancy; Chatterjee, Samit; Lisok, Ala; Gabrielson, Kathleen L.; Nimmagadda, Sridhar; Pomper, Martin G.; Foster, F. Stuart

2017-01-01

Prostate specific membrane antigen (PSMA) targeted microbubbles (MBs) were developed using bioorthogonal chemistry. Streptavidin-labeled MBs were treated with a biotinylated tetrazine (MBTz) and targeted to PSMA expressing cells using trans-cyclooctene (TCO)-functionalized anti-PSMA antibodies (TCO-anti-PSMA). The extent of MB binding to PSMA positive cells for two different targeting strategies was determined using an in vitro flow chamber. The initial approach involved pretargeting, where TCO-anti-PSMA was first incubated with PSMA expressing cells and followed by MBTz, which subsequently showed a 2.8 fold increase in the number of bound MBs compared to experiments performed in the absence of TCO-anti-PSMA. Using direct targeting, where TCO-anti-PSMA was linked to MBTz prior to initiation of the assay, a 5-fold increase in binding compared to controls was observed. The direct targeting approach was subsequently evaluated in vivo using a human xenograft tumor model and two different PSMA-targeting antibodies. The US signal enhancements observed were 1.6- and 5.9-fold greater than that for non-targeted MBs. The lead construct was also evaluated in a head-to-head study using mice bearing both PSMA positive or negative tumors in separate limbs. The human PSMA expressing tumors exhibited a 2-fold higher US signal compared to those tumors deficient in human PSMA. The results demonstrate both the feasibility of preparing PSMA-targeted MBs and the benefits of using bioorthogonal chemistry to create targeted US probes. PMID:28472168

Alkylating chemotherapy may exert a uniquely deleterious effect upon neo-antigen-targeting anticancer vaccination

PubMed Central

Litterman, Adam J; Dudek, Arkadiusz Z; Largaespada, David A

2013-01-01

Alkylating chemotherapy exerts both antineoplastic and immunostimulatory effects. However, in addition to depleting regulatory T cells (Treg), alkylating agents also mediate a long lasting antiproliferative effect on responder lymphocytes. Our recent findings indicate that this antiproliferative effect profoundly impairs vaccination-induced immune responses, especially in the case of vaccines that target specific tumor-associated neo-antigens that do not require Treg depletion. PMID:24251080

Liposomes-coated gold nanocages with antigens and adjuvants targeted delivery to dendritic cells for enhancing antitumor immune response.

PubMed

Liang, Ruijing; Xie, Jun; Li, Jun; Wang, Ke; Liu, Liping; Gao, Yujie; Hussain, Mubashir; Shen, Guanxin; Zhu, Jintao; Tao, Juan

2017-12-01

For nanovaccine-based cancer immunotherapy, dendritic cells (DCs) are one of the most powerful antigen presenting cells (APCs) that initiate and promote the maturation of antigen-specific cytotoxic T lymphocytes (e.g., CD8 + T cells) to induce the local and systemic antitumor immunity and further suppress the tumor metastasis and produce long-term protection against tumor. Thus, the activation and maturation of DCs is the prerequisite for efficient CD8 + T cell-based antitumor immune responses, which is considered as a primary and promising task for nanovaccine engineering. Herein, we introduce a versatile nanovaccine of liposomes-coated gold nanocages (Lipos-AuNCs) modified with DCs specific antibody aCD11c for targeted delivery of adjuvant MPLA and melanoma antigen peptide TRP2 to promote the activation and maturation of DCs, and enhance tumor specific T lymphocytes responses. Moreover, AuNCs accumulation and AuNCs-engulfed DCs migration to regional lymph nodes (RLNs) became real-time visualization through in vivo fluorescence and photoacoustic (PA) imaging to monitor the immunity process. In vivo experimental results demonstrated that the targeted antigen/adjuvants-loaded AuNCs exhibited enhanced antitumor immune response to inhibit tumor growth and metastasis in both B16-F10 prophylactic and lung metastasis models, which may act as a promising nanoplatform for antitumor immunotherapy and in vivo tracking. Copyright © 2017 Elsevier Ltd. All rights reserved.

Detection of influenza antigenic variants directly from clinical samples using polyclonal antibody based proximity ligation assays

PubMed Central

Martin, Brigitte E.; Jia, Kun; Sun, Hailiang; Ye, Jianqiang; Hall, Crystal; Ware, Daphne; Wan, Xiu-Feng

2016-01-01

Identification of antigenic variants is the key to a successful influenza vaccination program. The empirical serological methods to determine influenza antigenic properties require viral propagation. Here a novel quantitative PCR-based antigenic characterization method using polyclonal antibody and proximity ligation assays, or so-called polyPLA, was developed and validated. This method can detect a viral titer that is less than 1000 TCID50/mL. Not only can this method differentiate between different HA subtypes of influenza viruses but also effectively identify antigenic drift events within the same HA subtype of influenza viruses. Applications in H3N2 seasonal influenza data showed that the results from this novel method are consistent with those from the conventional serological assays. This method is not limited to the detection of antigenic variants in influenza but also other pathogens. It has the potential to be applied through a large-scale platform in disease surveillance requiring minimal biosafety and directly using clinical samples. PMID:25546251

Protamine-based nanoparticles as new antigen delivery systems.

PubMed

González-Aramundiz, José Vicente; Peleteiro Olmedo, Mercedes; González-Fernández, África; Alonso Fernández, María José; Csaba, Noemi Stefánia

2015-11-01

The use of biodegradable nanoparticles as antigen delivery vehicles is an attractive approach to overcome the problems associated with the use of Alum-based classical adjuvants. Herein we report, the design and development of protamine-based nanoparticles as novel antigen delivery systems, using recombinant hepatitis B surface antigen as a model viral antigen. The nanoparticles, composed of protamine and a polysaccharide (hyaluronic acid or alginate), were obtained using a mild ionic cross-linking technique. The size and surface charge of the nanoparticles could be modulated by adjusting the ratio of the components. Prototypes with optimal physicochemical characteristics and satisfactory colloidal stability were selected for the assessment of their antigen loading capacity, antigen stability during storage and in vitro and in vivo proof-of-concept studies. In vitro studies showed that antigen-loaded nanoparticles induced the secretion of cytokines by macrophages more efficiently than the antigen in solution, thus indicating a potential adjuvant effect of the nanoparticles. Finally, in vivo studies showed the capacity of these systems to trigger efficient immune responses against the hepatitis B antigen following intramuscular administration, suggesting the potential interest of protamine-polysaccharide nanoparticles as antigen delivery systems. Copyright © 2015 Elsevier B.V. All rights reserved.

Virus-mimetic nanovesicles as a versatile antigen-delivery system

PubMed Central

Zhang, Pengfei; Chen, Yixin; Zeng, Yun; Shen, Chenguang; Li, Rui; Guo, Zhide; Li, Shaowei; Zheng, Qingbing; Chu, Chengchao; Wang, Zhantong; Zheng, Zizheng; Tian, Rui; Ge, Shengxiang; Zhang, Xianzhong; Xia, Ning-Shao; Liu, Gang; Chen, Xiaoyuan

2015-01-01

It is a critically important challenge to rapidly design effective vaccines to reduce the morbidity and mortality of unexpected pandemics. Inspired from the way that most enveloped viruses hijack a host cell membrane and subsequently release by a budding process that requires cell membrane scission, we genetically engineered viral antigen to harbor into cell membrane, then form uniform spherical virus-mimetic nanovesicles (VMVs) that resemble natural virus in size, shape, and specific immunogenicity with the help of surfactants. Incubation of major cell membrane vesicles with surfactants generates a large amount of nano-sized uniform VMVs displaying the native conformational epitopes. With the diverse display of epitopes and viral envelope glycoproteins that can be functionally anchored onto VMVs, we demonstrate VMVs to be straightforward, robust and tunable nanobiotechnology platforms for fabricating antigen delivery systems against a wide range of enveloped viruses. PMID:26504197

Complete responses of relapsed lymphoma following genetic modification of tumor-antigen presenting cells and T-lymphocyte transfer.

PubMed

Bollard, Catherine M; Gottschalk, Stephen; Leen, Ann M; Weiss, Heidi; Straathof, Karin C; Carrum, George; Khalil, Mariam; Wu, Meng-fen; Huls, M Helen; Chang, Chung-Che; Gresik, M Victoria; Gee, Adrian P; Brenner, Malcolm K; Rooney, Cliona M; Heslop, Helen E

2007-10-15

Epstein-Barr virus (EBV)-associated tumors developing in immunocompetent individuals present a challenge to immunotherapy, since they lack expression of immunodominant viral antigens. However, the tumors consistently express viral proteins including LMP2, which are immunologically "weak" but may nonetheless be targets for immune T cells. We previously showed that a majority of cytotoxic T lymphocytes (CTLs) reactivated using EBV-transformed B-lymphoblastoid cells lines (LCLs) contained minor populations of LMP2-specific T cells and homed to tumor sites. However, they did not produce remissions in patients with bulky disease. We have now used gene transfer into antigen-presenting cells (APCs) to augment the expression and immunogenicity of LMP2. These modified APCs increased the frequency of LMP2-specific CTLs by up to 100-fold compared with unmodified LCL-APCs. The LMP2-specific population expanded and persisted in vivo without adverse effects. Nine of 10 patients treated in remission of high-risk disease remain in remission, and 5 of 6 patients with active relapsed disease had a tumor response, which was complete in 4 and sustained for more than 9 months. It is therefore possible to generate immune responses to weak tumor antigens by ex vivo genetic modification of APCs and the CTLs so produced can have substantial antitumor activity. This study is registered at http://www.cancer.gov/clinicaltrials (protocol IDs: BCM-H-9936, NCT00062868, NCT00070226).

Specific Mutation of a Gammaherpesvirus-Expressed Antigen in Response to CD8 T Cell Selection In Vivo

PubMed Central

Loh, Joy; Popkin, Daniel L.; Droit, Lindsay; Braaten, Douglas C.; Zhao, Guoyan; Zhang, Xin; Vachharajani, Punit; Myers, Nancy; Hansen, Ted H.

2012-01-01

Herpesviruses are thought to be highly genetically stable, and their use as vaccine vectors has been proposed. However, studies of the human gammaherpesvirus, Epstein-Barr virus, have found viral isolates containing mutations in HLA class I-restricted epitopes. Using murine gammaherpesvirus 68 expressing ovalbumin (OVA), we examined the stability of a gammaherpesvirus antigenic locus under strong CD8 T cell selection in vivo. OVA-specific CD8 T cells selected viral isolates containing mutations in the OVA locus but minimal alterations in other genomic regions. Thus, a CD8 T cell response to a gammaherpesvirus-expressed antigen that is not essential for replication or pathogenesis can result in selective mutation of that antigen in vivo. This finding may have relevance for the use of herpesvirus vectors for chronic antigen expression in vivo. PMID:22171269

Synthesis and Structure-Activity Relationships of Small Molecule Inhibitors of the Simian Virus 40 T Antigen Oncoprotein, an Anti-Polyomaviral Target

PubMed Central

Gupta, Tushar; Seguin, Sandlin P.; Liang, Mary; Resnick, Lynn; Goldberg, Margot T.; Manos-Turvey, Alexandra; Pipas, James M.; Wipf, Peter; Brodsky, Jeffrey L.

2014-01-01

Polyomavirus infections are common and relatively benign in the general human population but can become pathogenic in immunosuppressed patients. Because most treatments for polyomavirus-associated diseases nonspecifically target DNA replication, existing treatments for polyomavirus infection possess undesirable side effects. However, all polyomaviruses express Large Tumor Antigen (T Ag), which is unique to this virus family and may serve as a therapeutic target. Previous screening of pyrimidinone-peptoid hybrid compounds identified MAL2-11B and a MAL2-11B tetrazole derivative as inhibitors of viral replication and T Ag ATPase activity (IC50 of ~20-50μM). To improve upon this scaffold and to develop a structure-activity relationship for this new class of antiviral agents, several iterative series of MAL2-11B derivatives were synthesized. The replacement of a flexible methylene chain linker with a benzyl group or, alternatively, the addition of an ortho-methyl substituent on the biphenyl side chain in MAL2-11B yielded analogs with modestly improved IC50s (~15 μM), which retained antiviral activity. After combining both structural motifs, a new lead compound was identified that inhibited T Ag ATPase activity with an IC50 of ~5 μM. We suggest that the knowledge gained from the structure-activity relationship and a further refinement cycle of the MAL2-11B scaffold will provide a specific, novel therapeutic treatment option for polyomavirus infections and their associated diseases. PMID:25440730

Antigen vehiculization particles based on the Z protein of Junin virus.

PubMed

Borio, Cristina S; Bilen, Marcos F; Argüelles, Marcelo H; Goñi, Sandra E; Iserte, Javier A; Glikmann, Graciela; Lozano, Mario E

2012-11-02

Arenavirus matrix protein Z plays an important role in virus budding and is able to generate enveloped virus-like-particles (VLPs) in absence of any other viral proteins. In these VLPs, Z protein is associated to the plasma membrane inner surface by its myristoyl residue. Budding induction and vesicle formation properties can be exploited to generate enveloped VLPs platform. These structures can be designed to carry specific antigen in the inner side or on the surface of VLPs.Vaccines based on VLPs are a highly effective type of subunit vaccines that mimic the overall structure of virus particles in absence of viral nucleic acid, being noninfectious.In this work we assayed the capacity of Junin Z protein to produce VLPs carrying the green fluorescent protein (eGFP), as a model antigen. In this report the Junin Z protein ability to produce VLPs from 293T cells and its capacity to deliver a specific antigen (eGFP) fused to Z was evaluated. Confocal microscopy showed a particular membrane bending in cells expressing Z and a spot welded distribution in the cytoplasm. VLPs were detected by TEM (transmission electron microscopy) and were purified from cell supernatant. The proteinase protection assay demonstrated the VLPs integrity and the absence of degradation of the fused antigen, thus indicating its internal localization. Finally, immunization of mice with purified VLPs produced high titres of anti-eGFP antibodies compared to the controls. It was proved that VLPs can be generated from cells transfected with a fusion Junin virus Z-eGFP protein in absence of any other viral protein, and the capacity of Z protein to support fusions at the C-terminal, without impairing its budding activity, allowing vehiculization of specific antigens into VLPs.

Targeting the adenosine 2A receptor enhances chimeric antigen receptor T cell efficacy

PubMed Central

Beavis, Paul A.; Henderson, Melissa A.; Giuffrida, Lauren; Mills, Jane K.; Sek, Kevin; Cross, Ryan S.; Davenport, Alexander J.; John, Liza B.; Mardiana, Sherly; Slaney, Clare Y.; Johnstone, Ricky W.; Trapani, Joseph A.; Stagg, John; Loi, Sherene; Kats, Lev; Gyorki, David; Kershaw, Michael H.; Darcy, Phillip K.

2017-01-01

Chimeric antigen receptor (CAR) T cells have been highly successful in treating hematological malignancies, including acute and chronic lymphoblastic leukemia. However, treatment of solid tumors using CAR T cells has been largely unsuccessful to date, partly because of tumor-induced immunosuppressive mechanisms, including adenosine production. Previous studies have shown that adenosine generated by tumor cells potently inhibits endogenous antitumor T cell responses through activation of adenosine 2A receptors (A2ARs). Herein, we have observed that CAR activation resulted in increased A2AR expression and suppression of both murine and human CAR T cells. This was reversible using either A2AR antagonists or genetic targeting of A2AR using shRNA. In 2 syngeneic HER2+ self-antigen tumor models, we found that either genetic or pharmacological targeting of the A2AR profoundly increased CAR T cell efficacy, particularly when combined with PD-1 blockade. Mechanistically, this was associated with increased cytokine production of CD8+ CAR T cells and increased activation of both CD8+ and CD4+ CAR T cells. Given the known clinical relevance of the CD73/adenosine pathway in several solid tumor types, and the initiation of phase I trials for A2AR antagonists in oncology, this approach has high translational potential to enhance CAR T cell efficacy in several cancer types. PMID:28165340

Point detection of bacterial and viral pathogens using oral samples

NASA Astrophysics Data System (ADS)

Malamud, Daniel

2008-04-01

Oral samples, including saliva, offer an attractive alternative to serum or urine for diagnostic testing. This is particularly true for point-of-use detection systems. The various types of oral samples that have been reported in the literature are presented here along with the wide variety of analytes that have been measured in saliva and other oral samples. The paper focuses on utilizing point-detection of infectious disease agents, and presents work from our group on a rapid test for multiple bacterial and viral pathogens by monitoring a series of targets. It is thus possible in a single oral sample to identify multiple pathogens based on specific antigens, nucleic acids, and host antibodies to those pathogens. The value of such a technology for detecting agents of bioterrorism at remote sites is discussed.

Co-stimulatory signaling determines tumor antigen sensitivity and persistence of CAR T cells targeting PSCA+ metastatic prostate cancer

PubMed Central

Priceman, Saul J.; Gerdts, Ethan A.; Tilakawardane, Dileshni; Kennewick, Kelly T.; Murad, John P.; Park, Anthony K.; Jeang, Brook; Yamaguchi, Yukiko; Urak, Ryan; Weng, Lihong; Chang, Wen-Chung; Wright, Sarah; Pal, Sumanta; Reiter, Robert E.; Brown, Christine E.; Forman, Stephen J.

2018-01-01

ABSTRACT Advancing chimeric antigen receptor (CAR)-engineered adoptive T cells for the treatment of solid cancers is a major focus in the field of immunotherapy, given impressive recent clinical responses in hematological malignancies. Prostate cancer may be amenable to T cell-based immunotherapy since several tumor antigens, including prostate stem-cell antigen (PSCA), are widely over-expressed in metastatic disease. While antigen selectivity of CARs for solid cancers is crucial, it is problematic due to the absence of truly restricted tumor antigen expression and potential safety concerns with “on-target off-tumor” activity. Here, we show that the intracellular co-stimulatory signaling domain can determine a CAR's sensitivity for tumor antigen expression. A 4-1BB intracellular co-stimulatory signaling domain in PSCA-CARs confers improved selectivity for higher tumor antigen density, reduced T cell exhaustion phenotype, and equivalent tumor killing ability compared to PSCA-CARs containing the CD28 co-stimulatory signaling domain. PSCA-CARs exhibit robust in vivo anti-tumor activity in patient-derived bone-metastatic prostate cancer xenograft models, and 4-1BB-containing CARs show superior T cell persistence and control of disease compared with CD28-containing CARs. Our study demonstrates the importance of co-stimulation in defining an optimal CAR T cell, and also highlights the significance of clinically relevant models in developing solid cancer CAR T cell therapies. PMID:29308300

Antigenic Differences between AS03 Adjuvanted Influenza A (H1N1) Pandemic Vaccines: Implications for Pandemrix-Associated Narcolepsy Risk

PubMed Central

Vaarala, Outi; Vuorela, Arja; Partinen, Markku; Baumann, Marc; Freitag, Tobias L.; Meri, Seppo; Saavalainen, Päivi; Jauhiainen, Matti; Soliymani, Rabah; Kirjavainen, Turkka; Olsen, Päivi; Saarenpää-Heikkilä, Outi; Rouvinen, Juha; Roivainen, Merja; Nohynek, Hanna; Jokinen, Jukka; Julkunen, Ilkka; Kilpi, Terhi

2014-01-01

Background Narcolepsy results from immune-mediated destruction of hypocretin secreting neurons in hypothalamus, however the triggers and disease mechanisms are poorly understood. Vaccine-attributable risk of narcolepsy reported so far with the AS03 adjuvanted H1N1 vaccination Pandemrix has been manifold compared to the AS03 adjuvanted Arepanrix, which contained differently produced H1N1 viral antigen preparation. Hence, antigenic differences and antibody response to these vaccines were investigated. Methods and Findings Increased circulating IgG-antibody levels to Pandemrix H1N1 antigen were found in 47 children with Pandemrix-associated narcolepsy when compared to 57 healthy children vaccinated with Pandemrix. H1N1 antigen of Arepanrix inhibited poorly these antibodies indicating antigenic difference between Arepanrix and Pandemrix. High-resolution gel electrophoresis quantitation and mass spectrometry identification analyses revealed higher amounts of structurally altered viral nucleoprotein (NP) in Pandemrix. Increased antibody levels to hemagglutinin (HA) and NP, particularly to detergent treated NP, was seen in narcolepsy. Higher levels of antibodies to NP were found in children with DQB1*06∶02 risk allele and in DQB1*06∶02 transgenic mice immunized with Pandemrix when compared to controls. Conclusions This work identified 1) higher amounts of structurally altered viral NP in Pandemrix than in Arepanrix, 2) detergent-induced antigenic changes of viral NP, that are recognized by antibodies from children with narcolepsy, and 3) increased antibody response to NP in association of DQB1*06∶02 risk allele of narcolepsy. These findings provide a link between Pandemrix and narcolepsy. Although detailed mechanisms of Pandemrix in narcolepsy remain elusive, our results move the focus from adjuvant(s) onto the H1N1 viral proteins. PMID:25501681

Hepatitis B virus core antigen: synthesis in Escherichia coli and application in diagnosis.

PubMed Central

Stahl, S; MacKay, P; Magazin, M; Bruce, S A; Murray, K

1982-01-01

Fragments of hepatitis B virus DNA cloned in plasmid pBR322 carrying the gene for the viral core antigen have been placed under the control of the lac promoter of Escherichia coli. Several of the new recombinants direct higher levels of synthesis of the antigen, but the degree of enhancement varies with the different structures of the plasmids and hence the mRNAs produced. The antigen in crude bacterial lysates is a satisfactory diagnostic reagent for antibodies to the core antigen in serum samples. Images PMID:7041126

Chloroplast targeting of FanC, the major antigenic subunit of Escherichia coli K99 fimbriae, in transgenic soybean.

PubMed

Garg, Renu; Tolbert, Melanie; Oakes, Judy L; Clemente, Thomas E; Bost, Kenneth L; Piller, Kenneth J

2007-07-01

Enterotoxigenic Escherichia coli (ETEC) strains are a major cause of enteric diseases affecting livestock and humans. Edible transgenic plants producing E. coli fimbrial subunit proteins have the potential to vaccinate against these diseases, but have not reached their full potential as a renewable source of oral vaccines due in part to insufficient levels of recombinant protein accumulation. Previously, we reported that cytosol targeting of the E. coli K99 fimbrial subunit antigen resulted in FanC accumulation to approximately 0.4% of total soluble protein in soybean leaves (Piller et al. in Planta 222:6-18, 2005). In this study, we report on the subcellular targeting of FanC to chloroplasts. Twenty-two transgenic T1 progeny derived from seven individual T0 transformation events were characterized, and 17 accumulated transgenic FanC. All of the characterized events displayed relatively low T-DNA complexity, and all exhibited proper targeting of FanC to the chloroplast. Accumulation of chloroplast-targeted FanC was approximately 0.08% of total soluble leaf protein, or approximately 5-fold less than cytosol-targeted FanC. Protein analysis of leaves at various stages of maturity suggested stability of chloroplast-targeted FanC throughout leaf maturation. Furthermore, mice immunized intraperitoneally with protein extract derived from transgenic leaves expressing chloroplast-targeted FanC developed significant antibody titers against FanC. This is the first report of subcellular targeting of a vaccine subunit antigen in soybean.

Clustered Integrin Ligands as a Novel Approach for the Targeting of Non-Viral Vectors

NASA Astrophysics Data System (ADS)

Ng, Quinn Kwan Tai

Gene transfer or gene delivery is described as the process in which foreign DNA is introduced into cells. Over the years, gene delivery has gained the attention of many researchers and has been developed as powerful tools for use in biotechnology and medicine. With the completion of the Human Genome Project, such advances in technology allowed for the identification of diseases ranging from hereditary disorders to acquired ones (cancer) which were thought to be incurable. Gene therapy provides the means necessary to treat or eliminate genetic diseases from its origin, unlike traditional medicine which only treat symptoms. With ongoing clinical trials for gene therapy increasing, the greatest difficulty still lies in developing safe systems which can target cells of interest to provide efficient delivery. Nature, over millions of years of evolution, has provided an example of one of the most efficient delivery systems: viruses. Although the use of viruses for gene delivery has been well studied, the safety issues involving immunogenicity, insertional mutagenesis, high cost, and poor reproducibility has provided problems for their clinical application. From understanding viruses, we gain insight to designing new systems for non-viral gene delivery. One of these techniques utilized by adenoviruses is the clustering of ligands on its surface through the use of a protein called a penton base. Through the use of nanotechnology we can mimic this basic concept in non-viral gene delivery systems. This dissertation research is focused on developing and applying a novel system for displaying the integrin binding ligand (RGD) in a constrained manner to form a clustered integrin ligand binding platform to be used to enhance the targeting and efficiency of non-viral gene delivery vectors. Peptide mixed monolayer protected gold nanoparticles provides a suitable surface for ligand clustering. A relationship between the peptide ratios in the reaction solution used to form these

Induction of Immune Tolerance to Foreign Protein via Adeno-Associated Viral Vector Gene Transfer in Mid-Gestation Fetal Sheep

PubMed Central

Davey, Marcus G.; Riley, John S.; Andrews, Abigail; Tyminski, Alec; Limberis, Maria; Pogoriler, Jennifer E.; Partridge, Emily; Olive, Aliza; Hedrick, Holly L.; Flake, Alan W.; Peranteau, William H.

2017-01-01

A major limitation to adeno-associated virus (AAV) gene therapy is the generation of host immune responses to viral vector antigens and the transgene product. The ability to induce immune tolerance to foreign protein has the potential to overcome this host immunity. Acquisition and maintenance of tolerance to viral vector antigens and transgene products may also permit repeat administration thereby enhancing therapeutic efficacy. In utero gene transfer (IUGT) takes advantage of the immunologic immaturity of the fetus to induce immune tolerance to foreign antigens. In this large animal study, in utero administration of AAV6.2, AAV8 and AAV9 expressing green fluorescent protein (GFP) to ~60 day fetal sheep (term: ~150 days) was performed. Transgene expression and postnatal immune tolerance to GFP and viral antigens were assessed. We demonstrate 1) hepatic expression of GFP 1 month following in utero administration of AAV6.2.GFP and AAV8.GFP, 2) in utero recipients of either AAV6.2.GFP or AAV8.GFP fail to mount an anti-GFP antibody response following postnatal GFP challenge and lack inflammatory cellular infiltrates at the intramuscular site of immunization, 3) a serotype specific anti-AAV neutralizing antibody response is elicited following postnatal challenge of in utero recipients of AAV6.2 or AAV8 with the corresponding AAV serotype, and 4) durable hepatic GFP expression was observed up to 6 months after birth in recipients of AAV8.GFP but expression was lost between 1 and 6 months of age in recipients of AAV6.2.GFP. The current study demonstrates, in a preclinical large animal model, the potential of IUGT to achieve host immune tolerance to the viral vector transgene product but also suggests that a single exposure to the vector capsid proteins at the time of IUGT is inadequate to induce tolerance to viral vector antigens. PMID:28141818

Bayesian nonparametric clustering in phylogenetics: modeling antigenic evolution in influenza.

PubMed

Cybis, Gabriela B; Sinsheimer, Janet S; Bedford, Trevor; Rambaut, Andrew; Lemey, Philippe; Suchard, Marc A

2018-01-30

Influenza is responsible for up to 500,000 deaths every year, and antigenic variability represents much of its epidemiological burden. To visualize antigenic differences across many viral strains, antigenic cartography methods use multidimensional scaling on binding assay data to map influenza antigenicity onto a low-dimensional space. Analysis of such assay data ideally leads to natural clustering of influenza strains of similar antigenicity that correlate with sequence evolution. To understand the dynamics of these antigenic groups, we present a framework that jointly models genetic and antigenic evolution by combining multidimensional scaling of binding assay data, Bayesian phylogenetic machinery and nonparametric clustering methods. We propose a phylogenetic Chinese restaurant process that extends the current process to incorporate the phylogenetic dependency structure between strains in the modeling of antigenic clusters. With this method, we are able to use the genetic information to better understand the evolution of antigenicity throughout epidemics, as shown in applications of this model to H1N1 influenza. Copyright © 2017 John Wiley & Sons, Ltd. Copyright © 2017 John Wiley & Sons, Ltd.

Asymmetric Arginine dimethylation of Epstein-Barr virus nuclear antigen 2 promotes DNA targeting

DOE Office of Scientific and Technical Information (OSTI.GOV)

Gross, Henrik; Barth, Stephanie; Palermo, Richard D.

The Epstein-Barr virus (EBV) growth-transforms B-lymphocytes. The virus-encoded nuclear antigen 2 (EBNA2) is essential for transformation and activates gene expression by association with DNA-bound transcription factors such as RBPJkappa (CSL/CBF1). We have previously shown that EBNA2 contains symmetrically dimethylated Arginine (sDMA) residues. Deletion of the RG-repeat results in a reduced ability of the virus to immortalise B-cells. We now show that the RG repeat also contains asymmetrically dimethylated Arginines (aDMA) but neither non-methylated (NMA) Arginines nor citrulline residues. We demonstrate that only aDMA-containing EBNA2 is found in a complex with DNA-bound RBPJkappa in vitro and preferentially associates with the EBNA2-responsivemore » EBV C, LMP1 and LMP2A promoters in vivo. Inhibition of methylation in EBV-infected cells results in reduced expression of the EBNA2-regulated viral gene LMP1, providing additional evidence that methylation is a prerequisite for DNA-binding by EBNA2 via association with the transcription factor RBPJkappa.« less

Ex vivo screening for immunodominant viral epitopes by quantitative real time polymerase chain reaction (qRT-PCR)

PubMed Central

Provenzano, Maurizio; Mocellin, Simone; Bonginelli, Paola; Nagorsen, Dirk; Kwon, Seog-Woon; Stroncek, David

2003-01-01

The identification and characterization of viral epitopes across the Human Leukocyte Antigen (HLA) polymorphism is critical for the development of actives-specific or adoptive immunotherapy of virally-mediated diseases. This work investigates whether cytokine mRNA transcripts could be used to identify epitope-specific HLA-restricted memory T lymphocytes reactivity directly in fresh peripheral blood mononuclear cells (PBMCs) from viral-seropositive individuals in response to ex vivo antigen recall. PBMCs from HLA-A*0201 healthy donors, seropositive for Cytomegalovirus (CMV) and Influenza (Flu), were exposed for different periods and at different cell concentrations to the HLA-A*0201-restricted viral FluM158–66 and CMVpp65495–503 peptides. Quantitative real time PCR (qRT-PCR) was employed to evaluate memory T lymphocyte immune reactivation by measuring the production of mRNA encoding four cytokines: Interferon-γ (IFN-γ), Interleukin-2 (IL-2), Interleukin-4 (IL-4), and Interleukin-10 (IL-10). We could characterize cytokine expression kinetics that illustrated how cytokine mRNA levels could be used as ex vivo indicators of T cell reactivity. Particularly, IFN-γ mRNA transcripts could be consistently detected within 3 to 12 hours of short-term stimulation in levels sufficient to screen for HLA-restricted viral immune responses in seropositive subjects. This strategy will enhance the efficiency of the identification of viral epitopes independently of the individual HLA phenotype and could be used to follow the intensity of immune responses during disease progression or in response to in vivo antigen-specific immunization. PMID:14675481

Dual CD19 and CD123 targeting prevents antigen-loss relapses after CD19-directed immunotherapies

PubMed Central

Barrett, David M.; Shestova, Olga; Hofmann, Ted J.; Perazzelli, Jessica; Klichinsky, Michael; Aikawa, Vania; Nazimuddin, Farzana; Kozlowski, Miroslaw; Scholler, John; Lacey, Simon F.; Melenhorst, Jan J.; Morrissette, Jennifer J.D.; Christian, David A.; Hunter, Christopher A.; Kalos, Michael; Porter, David L.; June, Carl H.; Grupp, Stephan A.

2016-01-01

Potent CD19-directed immunotherapies, such as chimeric antigen receptor T cells (CART) and blinatumomab, have drastically changed the outcome of patients with relapsed/refractory B cell acute lymphoblastic leukemia (B-ALL). However, CD19-negative relapses have emerged as a major problem that is observed in approximately 30% of treated patients. Developing approaches to preventing and treating antigen-loss escapes would therefore represent a vertical advance in the field. Here, we found that in primary patient samples, the IL-3 receptor α chain CD123 was highly expressed on leukemia-initiating cells and CD19-negative blasts in bulk B-ALL at baseline and at relapse after CART19 administration. Using intravital imaging in an antigen-loss CD19-negative relapse xenograft model, we determined that CART123, but not CART19, recognized leukemic blasts, established protracted synapses, and eradicated CD19-negative leukemia, leading to prolonged survival. Furthermore, combining CART19 and CART123 prevented antigen-loss relapses in xenograft models. Finally, we devised a dual CAR-expressing construct that combined CD19- and CD123-mediated T cell activation and demonstrated that it provides superior in vivo activity against B-ALL compared with single-expressing CART or pooled combination CART. In conclusion, these findings indicate that targeting CD19 and CD123 on leukemic blasts represents an effective strategy for treating and preventing antigen-loss relapses occurring after CD19-directed therapies PMID:27571406

ESAT-6 Targeting to DEC205+ Antigen Presenting Cells Induces Specific-T Cell Responses against ESAT-6 and Reduces Pulmonary Infection with Virulent Mycobacterium tuberculosis.

PubMed

Silva-Sánchez, Aarón; Meza-Pérez, Selene; Flores-Langarica, Adriana; Donis-Maturano, Luis; Estrada-García, Iris; Calderón-Amador, Juana; Hernández-Pando, Rogelio; Idoyaga, Juliana; Steinman, Ralph M; Flores-Romo, Leopoldo

2015-01-01

Airways infection with Mycobacterium tuberculosis (Mtb) is contained mostly by T cell responses, however, Mtb has developed evasion mechanisms which affect antigen presenting cell (APC) maturation/recruitment delaying the onset of Ag-specific T cell responses. Hypothetically, bypassing the natural infection routes by delivering antigens directly to APCs may overcome the pathogen's naturally evolved evasion mechanisms, thus facilitating the induction of protective immune responses. We generated a murine monoclonal fusion antibody (α-DEC-ESAT) to deliver Early Secretory Antigen Target (ESAT)-6 directly to DEC205+ APCs and to assess its in vivo effects on protection associated responses (IFN-γ production, in vivo CTL killing, and pulmonary mycobacterial load). Treatment with α-DEC-ESAT alone induced ESAT-6-specific IFN-γ producing CD4+ T cells and prime-boost immunization prior to Mtb infection resulted in early influx (d14 post-infection) and increased IFN-γ+ production by specific T cells in the lungs, compared to scarce IFN-γ production in control mice. In vivo CTL killing was quantified in relevant tissues upon transferring target cells loaded with mycobacterial antigens. During infection, α-DEC-ESAT-treated mice showed increased target cell killing in the lungs, where histology revealed cellular infiltrate and considerably reduced bacterial burden. Targeting the mycobacterial antigen ESAT-6 to DEC205+ APCs before infection expands specific T cell clones responsible for early T cell responses (IFN-γ production and CTL activity) and substantially reduces lung bacterial burden. Delivering mycobacterial antigens directly to APCs provides a unique approach to study in vivo the role of APCs and specific T cell responses to assess their potential anti-mycobacterial functions.

ESAT-6 Targeting to DEC205+ Antigen Presenting Cells Induces Specific-T Cell Responses against ESAT-6 and Reduces Pulmonary Infection with Virulent Mycobacterium tuberculosis

PubMed Central

Silva-Sánchez, Aarón; Meza-Pérez, Selene; Flores-Langarica, Adriana; Donis-Maturano, Luis; Estrada-García, Iris; Calderón-Amador, Juana; Hernández-Pando, Rogelio; Idoyaga, Juliana; Flores-Romo, Leopoldo

2015-01-01

Airways infection with Mycobacterium tuberculosis (Mtb) is contained mostly by T cell responses, however, Mtb has developed evasion mechanisms which affect antigen presenting cell (APC) maturation/recruitment delaying the onset of Ag-specific T cell responses. Hypothetically, bypassing the natural infection routes by delivering antigens directly to APCs may overcome the pathogen’s naturally evolved evasion mechanisms, thus facilitating the induction of protective immune responses. We generated a murine monoclonal fusion antibody (α-DEC-ESAT) to deliver Early Secretory Antigen Target (ESAT)-6 directly to DEC205+ APCs and to assess its in vivo effects on protection associated responses (IFN-γ production, in vivo CTL killing, and pulmonary mycobacterial load). Treatment with α-DEC-ESAT alone induced ESAT-6-specific IFN-γ producing CD4+ T cells and prime-boost immunization prior to Mtb infection resulted in early influx (d14 post-infection) and increased IFN-γ+ production by specific T cells in the lungs, compared to scarce IFN-γ production in control mice. In vivo CTL killing was quantified in relevant tissues upon transferring target cells loaded with mycobacterial antigens. During infection, α-DEC-ESAT-treated mice showed increased target cell killing in the lungs, where histology revealed cellular infiltrate and considerably reduced bacterial burden. Targeting the mycobacterial antigen ESAT-6 to DEC205+ APCs before infection expands specific T cell clones responsible for early T cell responses (IFN-γ production and CTL activity) and substantially reduces lung bacterial burden. Delivering mycobacterial antigens directly to APCs provides a unique approach to study in vivo the role of APCs and specific T cell responses to assess their potential anti-mycobacterial functions. PMID:25915045

Hepatitis B antigen and viral hepatitis type B in India*

PubMed Central

John, T. Jacob; Carman, Robert H.; Hill, Peter G.

1974-01-01

Surveys were conducted to determine the occurrence of overt hepatitis manifested by jaundice in groups of hospital patients who had survived their initial illness. Of those who had been given blood that had not been screened for the presence of hepatitis B antigen (HB Ag) 9.4% reported that they had had jaundice during the 6 months following transfusion. During a similar period jaundice was reported by only 1.1% of those who had received blood found to be negative for HB Ag by the cross-over electrophoresis test. This difference is highly significant. Jaundice was reported by 25.5% of recipients of HB Ag-positive blood. Of a control population of hospitalized but non-transfused patients 2.2% reported jaundice within 6 months of hospitalization. The risk of overt hepatitis from HB Ag in transfused blood is high in southern India and is similar to that in temperate countries where the occurrence of antigen carriers, and presumably of immune status, are lower than in the tropics. PMID:4549611

How single mutations affect viral escape from broad and narrow antibodies to H1 influenza hemagglutinin.

PubMed

Doud, Michael B; Lee, Juhye M; Bloom, Jesse D

2018-04-11

Influenza virus can escape most antibodies with single mutations. However, rare antibodies broadly neutralize many viral strains. It is unclear how easily influenza virus might escape such antibodies if there was strong pressure to do so. Here, we map all single amino-acid mutations that increase resistance to broad antibodies to H1 hemagglutinin. Our approach not only identifies antigenic mutations but also quantifies their effect sizes. All antibodies select mutations, but the effect sizes vary widely. The virus can escape a broad antibody to hemagglutinin's receptor-binding site the same way it escapes narrow strain-specific antibodies: via single mutations with huge effects. In contrast, broad antibodies to hemagglutinin's stalk only select mutations with small effects. Therefore, among the antibodies we examine, breadth is an imperfect indicator of the potential for viral escape via single mutations. Antibodies targeting the H1 hemagglutinin stalk are quantifiably harder to escape than the other antibodies tested here.

Analysis of HSV viral reactivation in explants of sensory neurons

PubMed Central

Turner, Anne-Marie W.; Kristie, Thomas M.

2014-01-01

As with all Herpesviruses, Herpes simplex virus (HSV) has both a lytic replication phase and a latency-reactivation cycle. During lytic replication, there is an ordered cascade of viral gene expression that leads to the synthesis of infectious viral progeny. In contrast, latency is characterized by the lack of significant lytic gene expression and the absence of infectious virus. Reactivation from latency is characterized by the re-entry of the virus into the lytic replication cycle and the production of recurrent disease. This unit describes the establishment of the mouse sensory neuron model of HSV-1 latency-reactivation as a useful in vivo system for the analysis of mechanisms involved in latency and reactivation. Assays including the determination of viral yields, immunohistochemical/immunofluorescent detection of viral antigens, and mRNA quantitation are used in experiments designed to investigate the network of cellular and viral proteins regulating HSV-1 lytic infection, latency, and reactivation. PMID:25367271

Immunotherapy for B-Cell Neoplasms using T Cells expressing Chimeric Antigen Receptors

PubMed Central

Boulassel, Mohamed-Rachid; Galal, Ahmed

2012-01-01

Immunotherapy with T cells expressing chimeric antigen receptors (CAR) is being evaluated as a potential treatment for B-cell neoplasms. In recent clinical trials it has shown promising results. As the number of potential candidate antigens expands, the choice of suitable target antigens becomes more challenging to design studies and to assess optimal efficacy of CAR. Careful evaluation of candidate target antigens is required to ensure that T cells expressing CAR will preferentially kill malignant cells with a minimal toxicity against normal tissues. B cells express specific surface antigens that can theoretically act as targets for CAR design. Although many of these antigens can stimulate effective cellular immune responses in vivo, their implementation in clinical settings remains a challenge. Only targeted B-cell antigens CD19 and CD20 have been tested in clinical trials. This article reviews exploitable B cell surface antigens for CAR design and examines obstacles that could interfere with the identification of potentially useful cellular targets. PMID:23269948

Hsp70 enhances presentation of FMDV antigen to bovine CD4+ T cells in vitro

PubMed Central

McLaughlin, Kerry; Seago, Julian; Robinson, Lucy; Kelly, Charles; Charleston, Bryan

2010-01-01

Foot-and-mouth disease virus (FMDV) is the causative agent of a highly contagious acute vesicular disease affecting cloven-hoofed animals, including cattle, sheep and pigs. The current vaccine induces a rapid humoral response, but the duration of the protective antibody response is variable, possibly associated with a variable specific CD4+ T cell response. We investigated the use of heat shock protein 70 (Hsp70) as a molecular chaperone to target viral antigen to the Major Histocompatibility Complex (MHC) class II pathway of antigen presenting cells and generate enhanced MHC II-restricted CD4+ T cell responses in cattle. Monocytes and CD4+ T cells from FMDV vaccinated cattle were stimulated in vitro with complexes of Hsp70 and FMDV peptide, or peptide alone. Hsp70 was found to consistently improve the presentation of a 25-mer FMDV peptide to CD4+ T cells, as measured by T cell proliferation. Complex formation was required for the enhanced effects and Hsp70 alone did not stimulate proliferation. This study provides further evidence that Hsp70:peptide complexes can enhance antigen-specific CD4+ T cell responses in vitro for an important pathogen of livestock. PMID:20167197

Engineering Chimeric Antigen Receptors

PubMed Central

Kulemzin, S. V.; Kuznetsova, V. V.; Mamonkin, M.; Taranin, A. V.; Gorchakov, A. A.

2017-01-01

Chimeric antigen receptors (CARs) are recombinant protein molecules that redirect cytotoxic lymphocytes toward malignant and other target cells. The high feasibility of manufacturing CAR-modified lymphocytes for the therapy of cancer has spurred the development and optimization of new CAR T cells directed against a broad range of target antigens. In this review, we describe the main structural and functional elements constituting a CAR, discuss the roles of these elements in modulating the anti-tumor activity of CAR T cells, and highlight alternative approaches to CAR engineering. PMID:28461969

Cell-mediated immunity to herpes simplex virus: recognition of type-specific and type-common surface antigens by cytotoxic T cell populations.

PubMed Central

Eberle, R; Russell, R G; Rouse, B T

1981-01-01

In this communication, we examine the specificity of anti-herpes simplex virus (HSV) cytotoxic T lymphocytes (CTL). Serological studies of the two related HSV serotypes (HSV-1 and HSV-2) have revealed both type-specific and cross-reactive antigenic determinants in the viral envelope and on the surface of infected cells. By analysis of cytotoxicity of CTL, generated in vitro by restimulation of splenocytes from mice primed with one or the other HSV serotype, the recognition of both type-specific and cross-reactive determinants on infected target cells by anti-HSV CTL was detectable. Thus, effector cells generated by priming and restimulating with the same virus recognized both type-specific and cross-reactive determinants on target cells infected with the homologous virus, but only cross-reactive determinants on target cells infected with the heterologous HSV serotype. CTL generated by restimulation with the heterologous virus were capable of recognizing only the cross-reactive determinants on either HSV-1- or HSV-2-infected target cells. These results indicate that two subpopulations of CTL exist in a population of anti-HSV immune spleen cells--those which recognize type-specific determinants and those specific for cross-reactive antigenic determinants present on the surface of HSV infected cells. The type-specific subset of anti-HSV CTL was shown to recognize the gC glycoprotein of HSV-1 infected target cells. In addition to the gC glycoprotein, at least one other type-specific surface antigen was also recognized by anti-HSV CTL in addition to the cross-reactive determinants recognized by anti-HSV CTL. PMID:6277790

Sperm fibrous sheath proteins: a potential new class of target antigens for use in human therapeutic cancer vaccines

PubMed Central

Chiriva-Internati, Maurizio; Cobos, Everardo; Da Silva, Diane M.

2008-01-01

Cancer vaccines have been demonstrated to be a promising strategy for treating human neoplastic disease, but one of the limitations of these vaccines remains the paucity of target antigens to which to direct an effective immune response. We hypothesize that sperm fibrous sheath proteins may be a new class of useful antigens for developing successful cancer vaccines. This hypothesis is supported by the expression of two sperm fibrous sheath proteins, called sperm protein 17 and calcium-binding tyrosine-phosphorylation regulated protein, in tumors of unrelated histological origin and their capability to induce T cell-based immune responses. PMID:18433090

Antigenic Drift Defines a New D4 Subgenotype of Measles Virus.

PubMed

Muñoz-Alía, Miguel Ángel; Muller, Claude P; Russell, Stephen J

2017-06-01

The measles virus hemagglutinin (MeV-H) protein is the main target of protective neutralizing antibodies. Using a panel of monoclonal antibodies (MAbs) that recognize known major antigenic sites in MeV-H, we identified a D4 genotype variant that escapes neutralization by MAbs targeting the neutralizing epitope (NE) antigenic site. By site-directed mutagenesis, L249P was identified as the critical mutation disrupting the NE in this genotype D4 variant. Forty-two available D4 genotype gene sequences were subsequently analyzed and divided into 2 groups according to the presence or absence of the L249P MeV-H mutation. Further analysis of the MeV-N gene sequences of these 2 groups confirmed that they represent clearly definable, sequence-divergent D4 subgenotypes, which we named subgenotypes D4.1 and D4.2. The subgenotype D4.1 MeVs were isolated predominantly in Kenya and Ethiopia, whereas the MAb-resistant subgenotype D4.2 MeVs were isolated predominantly in France and Great Britain, countries with higher vaccine coverage rates. Interestingly, D4.2 subgenotype viruses showed a trend toward diminished susceptibility to neutralization by human sera pooled from approximately 60 to 80 North American donors. Escape from MAb neutralization may be a powerful epidemiological surveillance tool to monitor the evolution of new MeV subgenotypes. IMPORTANCE Measles virus is a paradigmatic RNA virus, as the antigenic composition of the vaccination has not needed to be updated since its discovery. The vaccine confers protection by inducing neutralizing antibodies that interfere with the function of the hemagglutinin protein. Viral strains are indistinguishable serologically, although characteristic nucleotide sequences differentiate 24 genotypes. In this work, we describe a distant evolutionary branch within genotype D4. Designated subgenotype D4.2, this virus is distinguishable by neutralization with vaccine-induced monoclonal antibodies that target the neutralizing epitope (NE

Antigenic Drift Defines a New D4 Subgenotype of Measles Virus

PubMed Central

Muller, Claude P.

2017-01-01

ABSTRACT The measles virus hemagglutinin (MeV-H) protein is the main target of protective neutralizing antibodies. Using a panel of monoclonal antibodies (MAbs) that recognize known major antigenic sites in MeV-H, we identified a D4 genotype variant that escapes neutralization by MAbs targeting the neutralizing epitope (NE) antigenic site. By site-directed mutagenesis, L249P was identified as the critical mutation disrupting the NE in this genotype D4 variant. Forty-two available D4 genotype gene sequences were subsequently analyzed and divided into 2 groups according to the presence or absence of the L249P MeV-H mutation. Further analysis of the MeV-N gene sequences of these 2 groups confirmed that they represent clearly definable, sequence-divergent D4 subgenotypes, which we named subgenotypes D4.1 and D4.2. The subgenotype D4.1 MeVs were isolated predominantly in Kenya and Ethiopia, whereas the MAb-resistant subgenotype D4.2 MeVs were isolated predominantly in France and Great Britain, countries with higher vaccine coverage rates. Interestingly, D4.2 subgenotype viruses showed a trend toward diminished susceptibility to neutralization by human sera pooled from approximately 60 to 80 North American donors. Escape from MAb neutralization may be a powerful epidemiological surveillance tool to monitor the evolution of new MeV subgenotypes. IMPORTANCE Measles virus is a paradigmatic RNA virus, as the antigenic composition of the vaccination has not needed to be updated since its discovery. The vaccine confers protection by inducing neutralizing antibodies that interfere with the function of the hemagglutinin protein. Viral strains are indistinguishable serologically, although characteristic nucleotide sequences differentiate 24 genotypes. In this work, we describe a distant evolutionary branch within genotype D4. Designated subgenotype D4.2, this virus is distinguishable by neutralization with vaccine-induced monoclonal antibodies that target the neutralizing epitope

Recognition of Antigen-Specific B Cell Receptors From Chronic Lymphocytic Leukemia Patients By Synthetic Antigen Surrogates

PubMed Central

Sarkar, Mohosin; Liu, Yun; Morimoto, Jumpei; Peng, Haiyong; Aquino, Claudio; Rader, Christoph; Chiorazzi, Nicholas

2014-01-01

In patients with chronic lymphocytic leukemia (CLL), a single neoplastic antigen-specific B cell accumulates and overgrows other B cells, leading to immune deficiency. CLL is often treated with drugs that ablate all B cells, leading to further weakening of humoral immunity, and a more focused therapeutic strategy capable of targeting only the pathogenic B cells would represent a significant advance. One approach to this would be to develop synthetic surrogates of the CLL antigens allowing differentiation of the CLL cells and healthy B cells in a patient. Here, we describe discovery of non-peptidic molecules capable of targeting antigen-specific B cell receptors with good affinity and selectivity using a combinatorial library screen. We demonstrate that our hit compounds act as synthetic antigen surrogates and recognize CLL cells and not healthy B cells. Additionally, we argue that the technology we developed can be used for discovery of other classes of antigen surrogates. PMID:25467125

Recognition of antigen-specific B-cell receptors from chronic lymphocytic leukemia patients by synthetic antigen surrogates.

PubMed

Sarkar, Mohosin; Liu, Yun; Morimoto, Jumpei; Peng, Haiyong; Aquino, Claudio; Rader, Christoph; Chiorazzi, Nicholas; Kodadek, Thomas

2014-12-18

In patients with chronic lymphocytic leukemia (CLL), a single neoplastic antigen-specific B cell accumulates and overgrows other B cells, leading to immune deficiency. CLL is often treated with drugs that ablate all B cells, leading to further weakening of humoral immunity, and a more focused therapeutic strategy capable of targeting only the pathogenic B cells would represent a significant advance. One approach to this would be to develop synthetic surrogates of the CLL antigens allowing differentiation of the CLL cells and healthy B cells in a patient. Here, we describe nonpeptidic molecules capable of targeting antigen-specific B cell receptors with good affinity and selectivity using a combinatorial library screen. We demonstrate that our hit compounds act as synthetic antigen surrogates and recognize CLL cells and not healthy B cells. Additionally, we argue that the technology we developed can be used to identify other classes of antigen surrogates. Copyright © 2014 Elsevier Ltd. All rights reserved.

AFM force measurements of the gp120-sCD4 and gp120 or CD4 antigen-antibody interactions

DOE Office of Scientific and Technical Information (OSTI.GOV)

Chen, Yong, E-mail: dr_yongchen@hotmail.com; Department of Microbiology and Immunology, University of Illinois at Chicago, Chicago, IL 60612; Zeng, Gucheng

2011-04-08

Highlights: {yields} The unbinding force of sCD4-gp120 interaction was 25.45 {+-} 20.46 pN. {yields} The unbinding force of CD4 antigen-antibody interaction was 51.22 {+-} 34.64 pN. {yields} The unbinding force of gp120 antigen-antibody interaction was 89.87 {+-} 44.63 pN. {yields} The interaction forces between various HIV inhibitors and the target molecules are significantly different. {yields} Functionalizing on AFM tip or substrate of an interaction pair caused different results. -- Abstract: Soluble CD4 (sCD4), anti-CD4 antibody, and anti-gp120 antibody have long been regarded as entry inhibitors in human immunodeficiency virus (HIV) therapy. However, the interactions between these HIV entry inhibitors andmore » corresponding target molecules are still poorly understood. In this study, atomic force microscopy (AFM) was utilized to investigate the interaction forces among them. We found that the unbinding forces of sCD4-gp120 interaction, CD4 antigen-antibody interaction, and gp120 antigen-antibody interaction were 25.45 {+-} 20.46, 51.22 {+-} 34.64, and 89.87 {+-} 44.63 pN, respectively, which may provide important mechanical information for understanding the effects of viral entry inhibitors on HIV infection. Moreover, we found that the functionalization of an interaction pair on AFM tip or substrate significantly influenced the results, implying that we must perform AFM force measurement and analyze the data with more caution.« less

Prostate-specific membrane antigen as a target for cancer imaging and therapy

PubMed Central

KIESS, A. P.; BANERJEE, S. R.; MEASE, R. C.; ROWE, S. P.; RAO, A.; FOSS, C. A.; CHEN, Y.; YANG, X.; CHO, S. Y.; NIMMAGADDA, S.; POMPER, M. G.

2016-01-01

The prostate-specific membrane antigen (PSMA) is a molecular target whose use has resulted in some of the most productive work toward imaging and treating prostate cancer over the past two decades. A wide variety of imaging agents extending from intact antibodies to low-molecular-weight compounds permeate the literature. In parallel there is a rapidly expanding pool of antibody-drug conjugates, radiopharmaceutical therapeutics, small-molecule drug conjugates, theranostics and nanomedicines targeting PSMA. Such productivity is motivated by the abundant expression of PSMA on the surface of prostate cancer cells and within the neovasculature of other solid tumors, with limited expression in most normal tissues. Animating the field is a variety of small-molecule scaffolds upon which the radionuclides, drugs, MR-detectable species and nanoparticles can be placed with relative ease. Among those, the urea-based agents have been most extensively leveraged, with expanding clinical use for detection and more recently for radiopharmaceutical therapy of prostate cancer, with surprisingly little toxicity. PSMA imaging of other cancers is also appearing in the clinical literature, and may overtake FDG for certain indications. Targeting PSMA may provide a viable alternative or first-line approach to managing prostate and other cancers. PMID:26213140

Diagnosis and treatment of viral diseases in recipients of allogeneic hematopoietic stem cell transplantation

PubMed Central

2013-01-01

Viral infections are important causes of morbidity and mortality after allogeneic stem cell hematopoietic transplantation (allo-HSCT). Although most viral infections present with asymptomatic or subclinical manifestations, viruses may result in fatal complications in severe immunocompromised recipients. Reactivation of latent viruses, such as herpesviruses, is frequent during the immunosuppression that occurs with allo-HSCT. Viruses acquired from community, such as the respiratory and gastrointestinal viruses, are also important pathogens of post-transplant viral diseases. Currently, molecular diagnostic methods have replaced or supplemented traditional methods, such as viral culture and antigen detection, in diagnosis of viral infections. The utilization of polymerase chain reaction facilitates the early diagnosis. In view of lacking efficacious agents for treatment of viral diseases, prevention of viral infections is extremely valuable. Application of prophylactic strategies including preemptive therapy reduces viral infections and diseases. Adoptive cellular therapy for restoring virus-specific immunity is a promising method in the treatment of viral diseases. PMID:24341630

Cellular immunity to viral antigens limits E1-deleted adenoviruses for gene therapy.

PubMed Central

Yang, Y; Nunes, F A; Berencsi, K; Furth, E E; Gönczöl, E; Wilson, J M

1994-01-01

An important limitation that has emerged in the use of adenoviruses for gene therapy has been loss of recombinant gene expression that occurs concurrent with the development of pathology in the organ expressing the transgene. We have used liver-directed approaches to gene therapy in mice to study mechanisms that underlie the problems with transient expression and pathology that have characterized in vivo applications of first-generation recombinant adenoviruses (i.e., those deleted of E1a and E1b). Our data are consistent with the following hypothesis. Cells harboring the recombinant viral genome express the transgene as desired; however, low-level expression of viral genes also occurs. A virus-specific cellular immune response is stimulated that leads to destruction of the genetically modified hepatocytes, massive hepatitis, and repopulation of the liver with nontransgene-containing hepatocytes. These findings suggest approaches for improving recombinant adenoviruses that are based on further crippling the virus to limit expression of nondeleted viral genes. Images PMID:8183921

Understanding original antigenic sin in influenza with a dynamical system.

PubMed

Pan, Keyao

2011-01-01

Original antigenic sin is the phenomenon in which prior exposure to an antigen leads to a subsequent suboptimal immune response to a related antigen. Immune memory normally allows for an improved and rapid response to antigens previously seen and is the mechanism by which vaccination works. I here develop a dynamical system model of the mechanism of original antigenic sin in influenza, clarifying and explaining the detailed spin-glass treatment of original antigenic sin. The dynamical system describes the viral load, the quantities of healthy and infected epithelial cells, the concentrations of naïve and memory antibodies, and the affinities of naïve and memory antibodies. I give explicit correspondences between the microscopic variables of the spin-glass model and those of the present dynamical system model. The dynamical system model reproduces the phenomenon of original antigenic sin and describes how a competition between different types of B cells compromises the overall effect of immune response. I illustrate the competition between the naïve and the memory antibodies as a function of the antigenic distance between the initial and subsequent antigens. The suboptimal immune response caused by original antigenic sin is observed when the host is exposed to an antigen which has intermediate antigenic distance to a second antigen previously recognized by the host's immune system.

Enhanced cytotoxicity of natural killer cells following the acquisition of chimeric antigen receptors through trogocytosis.

PubMed

Cho, Fu-Nan; Chang, Tsung-Hsien; Shu, Chih-Wen; Ko, Ming-Chin; Liao, Shuen-Kuei; Wu, Kang-Hsi; Yu, Ming-Sun; Lin, Shyh-Jer; Hong, Ying-Chung; Chen, Chien-Hsun; Hung, Chien-Hui; Chang, Yu-Hsiang

2014-01-01

Natural killer (NK) cells have the capacity to target tumors and are ideal candidates for immunotherapy. Viral vectors have been used to genetically modify in vitro expanded NK cells to express chimeric antigen receptors (CARs), which confer cytotoxicity against tumors. However, use of viral transduction methods raises the safety concern of viral integration into the NK cell genome. In this study, we used trogocytosis as a non-viral method to modify NK cells for immunotherapy. A K562 cell line expressing high levels of anti-CD19 CARs was generated as a donor cell to transfer the anti-CD19 CARs onto NK cells via trogocytosis. Anti-CD19 CAR expression was observed in expanded NK cells after these cells were co-cultured for one hour with freeze/thaw-treated donor cells expressing anti-CD19 CARs. Immunofluorescence analysis confirmed the localization of the anti-CD19 CARs on the NK cell surface. Acquisition of anti-CD19 CARs via trogocytosis enhanced NK cell-mediated cytotoxicity against the B-cell acute lymphoblastic leukemia (B-ALL) cell lines and primary B-ALL cells derived from patients. To our knowledge, this is the first report that describes the increased cytotoxicity of NK cells following the acquisition of CARs via trogocytosis. This novel strategy could be a potential valuable therapeutic approach for the treatment of B-cell tumors.

Human cytomegalovirus microRNA miR-US4-1 inhibits CD8+ T cell response by targeting the aminopeptidase ERAP1

PubMed Central

Kim, Sungchul; Lee, Sanghyun; Shin, Jinwook; Kim, Youngkyun; Evnouchidou, Irini; Kim, Donghyun; Kim, Young-Kook; Kim, Young-Eui; Ahn, Jin-Hyun; Riddell, Stanley R.; Stratikos, Efstratios; Kim, V. Narry; Ahn, Kwangseog

2012-01-01

The major histocompatibility complex (MHC) class I molecules present peptides on the cell surface by CD8+ T cells, which is critical for killing of virally infected or transformed cells. Precursors of MHC class I-presented peptides are trimmed to mature epitopes by endoplasmic reticulum aminopeptidase 1 (ERAP1). The US2-US11 genomic region of human cytomegalovirus (HCMV) is dispensable for viral replication and harbors 3 microRNAs (miRNAs). We show here the HCMV miR-US4-1 specifically down-regulates ERAP1 expression during viral infection. Accordingly, the trimming of HCMV-derived peptides is inhibited, leading to reduced susceptibility of infected cells to HCMV-specific cytotoxic T lymphocytes (CTLs). Our findings reveal a novel viral miRNA-based CTL evasion mechanism that targets a key step in the MHC class I antigen-processing pathway. PMID:21892175

HLA-B*27 subtype specificity determines targeting and viral evolution of a hepatitis C virus-specific CD8+ T cell epitope.

PubMed

Nitschke, Katja; Barriga, Alejandro; Schmidt, Julia; Timm, Jörg; Viazov, Sergei; Kuntzen, Thomas; Kim, Arthur Y; Lauer, Georg M; Allen, Todd M; Gaudieri, Silvana; Rauch, Andri; Lange, Christian M; Sarrazin, Christoph; Eiermann, Thomas; Sidney, John; Sette, Alessandro; Thimme, Robert; López, Daniel; Neumann-Haefelin, Christoph

2014-01-01

HLA-B*27 is associated with spontaneous HCV genotype 1 clearance. HLA-B*27-restricted CD8+ T cells target three NS5B epitopes. Two of these epitopes are dominantly targeted in the majority of HLA-B*27+ patients. In chronic infection, viral escape occurs consistently in these two epitopes. The third epitope (NS5B2820) was dominantly targeted in an acutely infected patient. This was in contrast, however, to the lack of recognition and viral escape in the large majority of HLA-B*27+ patients. Here, we set out to determine the host factors contributing to selective targeting of this epitope. Four-digit HLA class I typing and viral sequence analyses were performed in 78 HLA-B*27+ patients with chronic HCV genotype 1 infection. CD8+ T cell analyses were performed in a subset of patients. In addition, HLA/peptide affinity was compared for HLA-B*27:02 and 05. The NS5B2820 epitope is only restricted by the HLA-B*27 subtype HLA-B*27:02 (that is frequent in Mediterranean populations), but not by the prototype HLA-B*27 subtype B*27:05. Indeed, the epitope is very dominant in HLA-B*27:02+ patients and is associated with viral escape mutations at the anchor position for HLA-binding in 12 out of 13 HLA-B*27:02+ chronically infected patients. The NS5B2820 epitope is immunodominant in the context of HLA-B*27:02, but is not restricted by other HLA-B*27 subtypes. This finding suggests an important role of HLA subtypes in the restriction of HCV-specific CD8+ responses. With minor HLA subtypes covering up to 39% of specific populations, these findings may have important implications for the selection of epitopes for global vaccines. Copyright © 2013 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved.

Inhibition of HIV-1 Maturation via Small-Molecule Targeting of the Amino-Terminal Domain in the Viral Capsid Protein.

PubMed

Wang, Weifeng; Zhou, Jing; Halambage, Upul D; Jurado, Kellie A; Jamin, Augusta V; Wang, Yujie; Engelman, Alan N; Aiken, Christopher

2017-05-01

The human immunodeficiency virus type 1 (HIV-1) capsid protein is an attractive therapeutic target, owing to its multifunctionality in virus replication and the high fitness cost of amino acid substitutions in capsids to HIV-1 infectivity. To date, small-molecule inhibitors have been identified that inhibit HIV-1 capsid assembly and/or impair its function in target cells. Here, we describe the mechanism of action of the previously reported capsid-targeting HIV-1 inhibitor, Boehringer-Ingelheim compound 1 (C1). We show that C1 acts during HIV-1 maturation to prevent assembly of a mature viral capsid. However, unlike the maturation inhibitor bevirimat, C1 did not significantly affect the kinetics or fidelity of Gag processing. HIV-1 particles produced in the presence of C1 contained unstable capsids that lacked associated electron density and exhibited impairments in early postentry stages of infection, most notably reverse transcription. C1 inhibited assembly of recombinant HIV-1 CA in vitro and induced aberrant cross-links in mutant HIV-1 particles capable of spontaneous intersubunit disulfide bonds at the interhexamer interface in the capsid lattice. Resistance to C1 was conferred by a single amino acid substitution within the compound-binding site in the N-terminal domain of the CA protein. Our results demonstrate that the binding site for C1 represents a new pharmacological vulnerability in the capsid assembly stage of the HIV-1 life cycle. IMPORTANCE The HIV-1 capsid protein is an attractive but unexploited target for clinical drug development. Prior studies have identified HIV-1 capsid-targeting compounds that display different mechanisms of action, which in part reflects the requirement for capsid function at both the efferent and afferent phases of viral replication. Here, we show that one such compound, compound 1, interferes with assembly of the conical viral capsid during virion maturation and results in perturbations at a specific protein

Diabetes tolerogenic vaccines targeting antigen-specific inflammation

PubMed Central

Geng, Shuang; Zhang, Huiyuan; Zhou, Xian; He, Yue; Zhang, Xiaoqian; Xie, Xiaoping; Li, Chaofan; He, Zhonghuai; Yu, Qingling; Zhong, Yiwei; Lowrie, Douglas B; Zheng, Guoxing; Wang, Bin

2015-01-01

Tolerance controls the magnitude of inflammation, and balance between beneficial and harmful effects of inflammation is crucial for organ function and survival. Inadequate tolerance leads to various inflammatory diseases. Antigen specific tolerance is ideal for inflammation control as alternative anti-inflammatory interventions are non-specific and consequently increase the risk of infection and tumorigenesis. With inherent antigen specificity, tolerogenic vaccines are potentially ideal for control of inflammation. Although the concept of tolerogenic vaccines is still in its infancy, tolerogenic mucosal vaccines and specific immuno-therapies have long been proven effective in pioneering examples. Now a body of evidence supporting the concept of tolerogenic vaccines has also accumulated. Here we comment on recent successes of the tolerogenic vaccine concept, present new evidence with a type 1 diabetes vaccine as an example and draw conclusions on the advantages and potential for inflammatory disease control at the bedside. PMID:25622092

A Rapid-Response Humoral Vaccine Platform Exploiting Pre-Existing Non-Cognate Populations of Anti-Vaccine or Anti-Viral CD4+ T Helper Cells to Confirm B Cell Activation.

PubMed

Hills, Thomas; Jakeman, Phillip G; Carlisle, Robert C; Klenerman, Paul; Seymour, Leonard W; Cawood, Ryan

2016-01-01

The need for CD4+ T cell responses to arise de novo following vaccination can limit the speed of B cell responses. Populations of pre-existing vaccine-induced or anti-viral CD4+ T cells recognising distinct antigens could be exploited to overcome this limitation. We hypothesise that liposomal vaccine particles encapsulating epitopes that are recognised, after processing and B cell MHCII presentation, by pre-existing CD4+ T cells will exploit this pre-existing T cell help and result in improved antibody responses to distinct target antigens displayed on the particle surface. Liposomal vaccine particles were engineered to display the malaria circumsporozoite (CSP) antigen on their surface, with helper CD4+ epitopes from distinct vaccine or viral antigens contained within the particle core, ensuring the B cell response is raised but focused against CSP. In vivo vaccination studies were then conducted in C57Bl/6 mice as models of either vaccine-induced pre-existing CD4+ T cell immunity (using ovalbumin-OVA) or virus-induced pre-existing CD4+ T cell immunity (murine cytomegalovirus-MCMV). Following the establishment of pre-existing by vaccination (OVA in the adjuvant TiterMax® Gold) or infection with MCMV, mice were administered CSP-coated liposomal vaccines containing the relevant OVA or MCMV core CD4+ T cell epitopes. In mice with pre-existing anti-OVA CD4+ T cell immunity, these vaccine particles elicited rapid, high-titre, isotype-switched CSP-specific antibody responses-consistent with the involvement of anti-OVA T helper cells in confirming activation of anti-CSP B cells. Responses were further improved by entrapping TLR9 agonists, combining humoral vaccination signals 'one', 'two' and 'three' within one particle. Herpes viruses can establish chronic infection and elicit significant, persistent cellular immune responses. We then demonstrate that this principle can be extended to re-purpose pre-existing anti-MCMV immunity to enhance anti-CSP vaccine responses

A Simple Proteomics-Based Approach to Identification of Immunodominant Antigens from a Complex Pathogen: Application to the CD4 T Cell Response against Human Herpesvirus 6B

PubMed Central

Becerra-Artiles, Aniuska; Dominguez-Amorocho, Omar; Stern, Lawrence J.; Calvo-Calle, J. Mauricio

2015-01-01

Most of humanity is chronically infected with human herpesvirus 6 (HHV-6), with viral replication controlled at least in part by a poorly characterized CD4 T cell response. Identification of viral epitopes recognized by CD4 T cells is complicated by the large size of the herpesvirus genome and a low frequency of circulating T cells responding to the virus. Here, we present an alternative to classical epitope mapping approaches used to identify major targets of the T cell response to a complex pathogen like HHV-6B. In the approach presented here, extracellular virus preparations or virus-infected cells are fractionated by SDS-PAGE, and eluted fractions are used as source of antigens to study cytokine responses in direct ex vivo T cell activation studies. Fractions inducing significant cytokine responses are analyzed by mass spectrometry to identify viral proteins, and a subset of peptides from these proteins corresponding to predicted HLA-DR binders is tested for IFN-γ production in seropositive donors with diverse HLA haplotypes. Ten HHV-6B viral proteins were identified as immunodominant antigens. The epitope-specific response to HHV-6B virus was complex and variable between individuals. We identified 107 peptides, each recognized by at least one donor, with each donor having a distinctive footprint. Fourteen peptides showed responses in the majority of donors. Responses to these epitopes were validated using in vitro expanded cells and naturally expressed viral proteins. Predicted peptide binding affinities for the eight HLA-DRB1 alleles investigated here correlated only modestly with the observed CD4 T cell responses. Overall, the response to the virus was dominated by peptides from the major capsid protein U57 and major antigenic protein U11, but responses to other proteins including glycoprotein H (U48) and tegument proteins U54 and U14 also were observed. These results provide a means to follow and potentially modulate the CD4 T-cell immune response to HHV-6

[Clinical benefit of HCV core antigen assay in patients receiving interferon and ribavirin combination therapy].

PubMed

Higashimoto, Makiko; Takahashi, Masahiko; Jokyu, Ritsuko; Saito, Hidetsugu

2006-02-01

A highly sensitive second generation HCV core antigen assay has recently been developed. We compared viral disappearance and kinetics data between commercially available core antigen assays, Lumipulse Ortho HCV Ag, and a quantitative HCV RNA PCR assay, Cobas Amplicor HCV Monitor Test, Version 2 to estimate the predictive benefit of sustained viral response (SVR) and non-SVR in 59 patients treated with interferon and ribavirin combination therapy. We found a good correlation between HCV core Ag and HCV RNA level regardless of genotype. Although the sensitivity of the core antigen assay was lower than PCR, the dynamic range was broader than that of the PCR assay, so that we did not need to dilute the samples in 59 patients. We detected serial decline of core Ag levels in 24 hrs, 7 days and 14 days after interferon combination therapy. The decline of core antigen levels was significant in SVR patients compared to non-SVR as well as in genotype 2a, 2b patients compared to 1b. Core antigen-negative on day 1 could predict all 10 SVR patients (PPV = 100%), whereas RNA-negative could predict 22 SVR out of 25 on day 14 (PPV = 88.0%). None of the patients who had detectable serum core antigen on day 14 became SVR(NPV = 100%), although NPV was 91.2% on RNA negativity. An easy, simple, low cost new HCV core antigen detecting system seems to be useful for assessing and monitoring IFN treatment for HCV.

Evaluation of Mycobacterium tuberculosis Early Secreted Antigenic Target 6 Recombinant Protein as a Diagnostic Marker in Skin Test.

PubMed

Moradi, Jale; Mosavari, Nader; Ebrahimi, Mahmoud; Arefpajohi, Reza; Tebianian, Majid

2015-02-01

Tuberculosis (TB) is the leading infectious disease in the developing world. Delayed-type hypersensitivity skin test diagnoses TB using tuberculin purified protein derivative (PPD), but this test is incapable of distinguishing Mycobacterium tuberculosis (MTB) infection from bacillus Calmette-Guérin (BCG) vaccination or an infection caused by nontuberculous mycobacteria (NTM). This study was performed to evaluate the use of recombinant early secretory antigenic target 6 (rESAT-6), a secretory protein found only in MTB, Mycobacterium bovis, and few other mycobacterial species, as a skin marker for MTB in guinea pigs. We prepared recombinant MTB ESAT-6 and evaluated its use as a specific antigen for MTB in guinea pigs. Our results show that the purified MTB rESAT-6 antigen is capable of inducing a positive reaction only in guinea pigs sensitized to MTB. No such reaction was observed in the animals sensitized to M. bovis, BCG vaccination, or NTM (Mycobacterium avium). Our study results confirm that the ESAT-6 antigen is more specific to MTB infection than PPD and could be used in more specific skin tests for detection of MTB in large animals and in humans.

Antibody-Independent Control of γ-Herpesvirus Latency via B Cell Induction of Anti-Viral T Cell Responses

PubMed Central

McClellan, Kelly B; Gangappa, Shivaprakash; Speck, Samuel H; Virgin, Herbert W.

2006-01-01

B cells can use antibody-dependent mechanisms to control latent viral infections. It is unknown whether this represents the sole function of B cells during chronic viral infection. We report here that hen egg lysozyme (HEL)-specific B cells can contribute to the control of murine γ-herpesvirus 68 (γHV68) latency without producing anti-viral antibody. HEL-specific B cells normalized defects in T cell numbers and proliferation observed in B cell−/− mice during the early phase of γHV68 latency. HEL-specific B cells also reversed defects in CD8 and CD4 T cell cytokine production observed in B cell−/− mice, generating CD8 and CD4 T cells necessary for control of latency. Furthermore, HEL-specific B cells were able to present virally encoded antigen to CD8 T cells. Therefore, B cells have antibody independent functions, including antigen presentation, that are important for control of γ-herpesvirus latency. Exploitation of this property of B cells may allow enhanced vaccine responses to chronic virus infection. PMID:16789842

Clinical report: Detection and management of bovine viral diarrhea virus Type 1b in a large dairy herd

USDA-ARS?s Scientific Manuscript database

Case Description: 1,081 newborn calves from a commercial dairy were tested for bovine viral diarrhea virus antigen by pooled RT-PCR as part of a screening program. Ear tissue from twenty six calves initially tested positive and 14 confirmed positive with antigen capture ELISA two weeks later (1.3...

Vaccine to control the viral infection of fish

DOEpatents

Leong, Jo-Ann C.

1994-10-11

Subunit vaccines and their use for immunizing fish against infection by viruses are disclosed. In particular, plasmid pG8 is constructed by joining, with the plasmid pUC8, DNA which encodes the glycoprotein of infectious hematopoietic necrosis virus (IHNV). E. coli cells are transformed by pG8, whereby pure viral antigen is produced to provide a vaccine for the control of IHNV in fish.

“Viral déjà vu” elicits organ-specific immune disease independent of reactivity to self

PubMed Central

Merkler, Doron; Horvath, Edit; Bruck, Wolfgang; Zinkernagel, Rolf M.; del la Torre, Juan Carlos; Pinschewer, Daniel D.

2006-01-01

Autoimmune diseases are often precipitated by viral infections. Yet our current understanding fails to explain how viruses trigger organ-specific autoimmunity despite thymic tolerance extending to many nonlymphohematopoietic self antigens. Additionally, a key epidemiological finding needs to be explained: In genetically susceptible individuals, early childhood infections seem to predispose them to multiple sclerosis (MS) or type 1 diabetes years or even decades before clinical onset. In the present work, we show that the innate immune system of neonatal mice was sufficient to eliminate an attenuated lymphocytic choriomeningitis virus (LCMV) from most tissues except for the CNS, where the virus persisted in neurons (predisposing virus). Virus-specific cytotoxic T cells (CTLs) were neither deleted nor sufficiently primed to cause disease, but they were efficiently triggered in adulthood upon WT LCMV infection (precipitating virus). This defined sequence of viral infections caused severe CNS inflammation that was histomorphologically reminiscent of rasmussen encephalitis, a fatal human autoimmune disease. Yet disease in mice was mediated by antiviral CTLs targeting an epitope shared by the precipitating virus and the predisposing virus persisting in neurons (déjà vu). Thus the concept of “viral déjà vu” demonstrates how 2 related but independently encountered viral infections can cause organ-specific immune disease without molecular mimicry of self and without breaking self tolerance. PMID:16604192

NEP: web server for epitope prediction based on antibody neutralization of viral strains with diverse sequences

PubMed Central

Chuang, Gwo-Yu; Liou, David; Kwong, Peter D.; Georgiev, Ivelin S.

2014-01-01

Delineation of the antigenic site, or epitope, recognized by an antibody can provide clues about functional vulnerabilities and resistance mechanisms, and can therefore guide antibody optimization and epitope-based vaccine design. Previously, we developed an algorithm for antibody-epitope prediction based on antibody neutralization of viral strains with diverse sequences and validated the algorithm on a set of broadly neutralizing HIV-1 antibodies. Here we describe the implementation of this algorithm, NEP (Neutralization-based Epitope Prediction), as a web-based server. The users must supply as input: (i) an alignment of antigen sequences of diverse viral strains; (ii) neutralization data for the antibody of interest against the same set of antigen sequences; and (iii) (optional) a structure of the unbound antigen, for enhanced prediction accuracy. The prediction results can be downloaded or viewed interactively on the antigen structure (if supplied) from the web browser using a JSmol applet. Since neutralization experiments are typically performed as one of the first steps in the characterization of an antibody to determine its breadth and potency, the NEP server can be used to predict antibody-epitope information at no additional experimental costs. NEP can be accessed on the internet at http://exon.niaid.nih.gov/nep. PMID:24782517

The oncogenic potential of BK-polyomavirus is linked to viral integration into the human genome.

PubMed

Kenan, Daniel J; Mieczkowski, Piotr A; Burger-Calderon, Raquel; Singh, Harsharan K; Nickeleit, Volker

2015-11-01

It has been suggested that BK-polyomavirus is linked to oncogenesis via high expression levels of large T-antigen in some urothelial neoplasms arising following kidney transplantation. However, a causal association between BK-polyomavirus, large T-antigen expression and oncogenesis has never been demonstrated in humans. Here we describe an investigation using high-throughput sequencing of tumour DNA obtained from an urothelial carcinoma arising in a renal allograft. We show that a novel BK-polyomavirus strain, named CH-1, is integrated into exon 26 of the myosin-binding protein C1 gene (MYBPC1) on chromosome 12 in tumour cells but not in normal renal cells. Integration of the BK-polyomavirus results in a number of discrete alterations in viral gene expression, including: (a) disruption of VP1 protein expression and robust expression of large T-antigen; (b) preclusion of viral replication; and (c) deletions in the non-coding control region (NCCR), with presumed alterations in promoter feedback loops. Viral integration disrupts one MYBPC1 gene copy and likely alters its expression. Circular episomal BK-polyomavirus gene sequences are not found, and the renal allograft shows no productive polyomavirus infection or polyomavirus nephropathy. These findings support the hypothesis that integration of polyomaviruses is essential to tumourigenesis. It is likely that dysregulation of large T-antigen, with persistent over-expression in non-lytic cells, promotes cell growth, genetic instability and neoplastic transformation. © 2015 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.

Anti-CD22-chimeric antigen receptors targeting B-cell precursor acute lymphoblastic leukemia.

PubMed

Haso, Waleed; Lee, Daniel W; Shah, Nirali N; Stetler-Stevenson, Maryalice; Yuan, Constance M; Pastan, Ira H; Dimitrov, Dimiter S; Morgan, Richard A; FitzGerald, David J; Barrett, David M; Wayne, Alan S; Mackall, Crystal L; Orentas, Rimas J

2013-02-14

Immune targeting of B-cell malignancies using chimeric antigen receptors (CARs) is a promising new approach, but critical factors impacting CAR efficacy remain unclear. To test the suitability of targeting CD22 on precursor B-cell acute lymphoblastic leukemia (BCP-ALL), lymphoblasts from 111 patients with BCP-ALL were assayed for CD22 expression and all were found to be CD22-positive, with median CD22 expression levels of 3500 sites/cell. Three distinct binding domains targeting CD22 were fused to various TCR signaling domains ± an IgG heavy chain constant domain (CH2CH3) to create a series of vector constructs suitable to delineate optimal CAR configuration. CARs derived from the m971 anti-CD22 mAb, which targets a proximal CD22 epitope demonstrated superior antileukemic activity compared with those incorporating other binding domains, and addition of a 4-1BB signaling domain to CD28.CD3 constructs diminished potency, whereas increasing affinity of the anti-CD22 binding motif, and extending the CD22 binding domain away from the membrane via CH2CH3 had no effect. We conclude that second-generation m971 mAb-derived anti-CD22 CARs are promising novel therapeutics that should be tested in BCP-ALL.

Viral haemorrhagic fever and vascular alterations.

PubMed

Aleksandrowicz, P; Wolf, K; Falzarano, D; Feldmann, H; Seebach, J; Schnittler, H

2008-02-01

Pathogenesis of viral haemorrhagic fever (VHF) is closely associated with alterations of the vascular system. Among the virus families causing VHF, filoviruses (Marburg and Ebola) are the most fatal, and will be focused on here. After entering the body, Ebola primarily targets monocytes/macrophages and dendritic cells. Infected dendritic cells are largely impaired in their activation potency, likely contributing to the immune suppression that occurs during filovirus infection. Monocytes/macrophages, however, immediately activate after viral contact and release reasonable amounts of cytokines that target the vascular system, particularly the endothelial cells. Some underlying molecular mechanisms such as alteration of the vascular endothelial cadherin/catenin complex, tyrosine phosphorylation, expression of cell adhesion molecules, tissue factor and the effect of soluble viral proteins released from infected cells to the blood stream will be discussed.

Hairpin RNA Targeting Multiple Viral Genes Confers Strong Resistance to Rice Black-Streaked Dwarf Virus.

PubMed

Wang, Fangquan; Li, Wenqi; Zhu, Jinyan; Fan, Fangjun; Wang, Jun; Zhong, Weigong; Wang, Ming-Bo; Liu, Qing; Zhu, Qian-Hao; Zhou, Tong; Lan, Ying; Zhou, Yijun; Yang, Jie

2016-05-11

Rice black-streaked dwarf virus (RBSDV) belongs to the genus Fijivirus in the family of Reoviridae and causes severe yield loss in rice-producing areas in Asia. RNA silencing, as a natural defence mechanism against plant viruses, has been successfully exploited for engineering virus resistance in plants, including rice. In this study, we generated transgenic rice lines harbouring a hairpin RNA (hpRNA) construct targeting four RBSDV genes, S1, S2, S6 and S10, encoding the RNA-dependent RNA polymerase, the putative core protein, the RNA silencing suppressor and the outer capsid protein, respectively. Both field nursery and artificial inoculation assays of three generations of the transgenic lines showed that they had strong resistance to RBSDV infection. The RBSDV resistance in the segregating transgenic populations correlated perfectly with the presence of the hpRNA transgene. Furthermore, the hpRNA transgene was expressed in the highly resistant transgenic lines, giving rise to abundant levels of 21-24 nt small interfering RNA (siRNA). By small RNA deep sequencing, the RBSDV-resistant transgenic lines detected siRNAs from all four viral gene sequences in the hpRNA transgene, indicating that the whole chimeric fusion sequence can be efficiently processed by Dicer into siRNAs. Taken together, our results suggest that long hpRNA targeting multiple viral genes can be used to generate stable and durable virus resistance in rice, as well as other plant species.

Antigen targeting to antigen-presenting cells enhances presentation to class II-restricted T lymphocytes.

PubMed Central

Scardino, A; Paroli, M; De Petrillo, G; Michel, M L; Barnaba, V

1994-01-01

Receptor-mediated uptake increases by several orders of magnitude the efficiency of APC to internalize Ag, and is stringently required for the Ag-presenting function of T lymphocytes due to their inability to take up Ag non-specifically. We have previously reported that hepatitis B envelope antigen (HBenvAg) can be internalized by T cells via transferrin receptor (TfR). To evaluate if Ag targeting to receptors expressed on APC could be an effective tool for promoting Ag uptake and presentation, we tested the capacity of activated T cells not expressing TfR to induce HBenvAg-specific T-cell responses when pulsed with a hybrid particle containing HBenvAg coupled to gp120 of human immunodeficiency virus (HIV), exploiting the ability of gp120 to bind to CD4 receptor. We found that CD4+/TfR- T cells pulsed either with the hybrid particle or peptide (S193-207) but not with S, L Ag, a recombinant form of HBenvAg, induced a specific proliferative response of a T-cell clone recognizing peptide (S193-207) of HBenvAg. The finding that the addition of anti-CD4 monoclonal antibody (mAb) before the pulsing of CD4+/TfR- T cells with the hybrid particle drastically blocked the specific T-cell response, together with the finding that CD8+/TfR- T cells were unable to serve as APC even if pulsed with this molecule, demonstrated that CD4 receptor was crucial for the HBenvAg internalization. On the other hand, HBenvAg presentation by CD4+/TfR+ T cells pulsed with the hybrid particle was inhibited only when both anti-CD4 and anti-TfR were added before the pulsing. These results suggest that Ag targeting to APC receptors may be usefully exploited to improve Ag-presentation efficiency in potential immunotherapeutic approaches. PMID:7907575

Merkel cell polyomavirus small T antigen induces genome instability by E3 ubiquitin ligase targeting.

PubMed

Kwun, H J; Wendzicki, J A; Shuda, Y; Moore, P S; Chang, Y

2017-12-07

The formation of a bipolar mitotic spindle is an essential process for the equal segregation of duplicated DNA into two daughter cells during mitosis. As a result of deregulated cellular signaling pathways, cancer cells often suffer a loss of genome integrity that might etiologically contribute to carcinogenesis. Merkel cell polyomavirus (MCV) small T (sT) oncoprotein induces centrosome overduplication, aneuploidy, chromosome breakage and the formation of micronuclei by targeting cellular ligases through a sT domain that also inhibits MCV large T oncoprotein turnover. These results provide important insight as to how centrosome number and chromosomal stability can be affected by the E3 ligase targeting capacity of viral oncoproteins such as MCV sT, which may contribute to Merkel cell carcinogenesis.

Prospects for new viral vaccines.

PubMed

Marmion, B P

1980-08-11

Animal virology has made outstanding contributions to preventive medicine by the development of vaccines for the control of infectious disease in man and animals. Cost-benefit analysis indicates substantial savings in health care costs from the control of diseases such as smallpox, poliomyelitis, yellow fever and measels. Areas for further development include vaccines for influenza (living, attenuated virus), the herpes group (varicella: cytomegalovirus), respiratory syncytial virus, rotavirus and hepatitis A, B, and non A/non B. The general options for vaccine formulation are discussed with particular emphasis on approaches with the use of viral genetics to 'tailor make' vaccine viruses with defined growth potential in laboratory systems, low pathogenicity, and defined antigens. Current progress with the development of an inactivated hepatitis B vaccine is reviewed as a case study in vaccine development. The impact of recent experiments in cloning hepatitis B virus DNA in E. coli on the production of a purified viral polypeptide vaccine is assessed.

Characterization of viral proteins of Oryctes baculovirus and comparison between two geographical isolates.

PubMed

Mohan, K S; Gopinathan, K P

1989-01-01

Bacilliform Oryctes baculovirus particles have been visualized in electron micrographs of midgut sections from virus infected Oryctes rhinoceros beetles. Morphologically the Indian isolate (Oryctes baculovirus, KI) resembled the previously reported Oryctes baculovirus, isolate PV505. The constituent proteins of baculovirus KI have been analysed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and by Western blots using polyclonal antibodies raised against the complete viral particles, as probes. A total of forty eight viral proteins have been identified. Fourteen viral proteins were located on the viral envelope. Among the proteins constituting the nucleocapsid, three were located internally within the capsid. A 23.5 kDa protein was tightly associated with viral DNA in the nucleocapsid core. Two envelope and seven capsid proteins of KI and PV505 revealed differences in SDS-PAGE profiles and glycosylation patterns. Immunoblotting of KI and PV505 proteins with anti KI antiserum demonstrated antigenic differences between the two viral isolates.

Effective Targeting of Multiple B-Cell Maturation Antigen-Expressing Hematological Malignances by Anti-B-Cell Maturation Antigen Chimeric Antigen Receptor T Cells.

PubMed

Friedman, Kevin M; Garrett, Tracy E; Evans, John W; Horton, Holly M; Latimer, Howard J; Seidel, Stacie L; Horvath, Christopher J; Morgan, Richard A

2018-05-01

B-cell maturation antigen (BCMA) expression has been proposed as a marker for the identification of malignant plasma cells in patients with multiple myeloma (MM). Nearly all MM tumor cells express BCMA, while normal tissue expression is restricted to plasma cells and a subset of mature B cells. Consistent BCMA expression was confirmed on MM biopsies (29/29 BCMA+), and it was further demonstrated that BCMA is expressed in a substantial number of lymphoma samples, as well as primary chronic lymphocytic leukemia B cells. To target BCMA using redirected autologous T cells, lentiviral vectors (LVV) encoding chimeric antigen receptors (CARs) were constructed with four unique anti-BCMA single-chain variable fragments, fused to the CD137 (4-1BB) co-stimulatory and CD3ζ signaling domains. One LVV, BB2121, was studied in detail, and BB2121 CAR-transduced T cells (bb2121) exhibited a high frequency of CAR + T cells and robust in vitro activity against MM cell lines, lymphoma cell lines, and primary chronic lymphocytic leukemia peripheral blood. Based on receptor quantification, bb2121 recognized tumor cells expressing as little as 222 BCMA molecules per cell. The in vivo pharmacology of anti-BCMA CAR T cells was studied in NSG mouse models of human MM, Burkitt lymphoma, and mantle cell lymphoma, where mice received a single intravenous administration of vehicle, control vector-transduced T cells, or anti-BCMA CAR-transduced T cells. In all models, the vehicle and control CAR T cells failed to inhibit tumor growth. In contrast, treatment with bb2121 resulted in rapid and sustained elimination of the tumors and 100% survival in all treatment models. Together, these data support the further development of anti-BCMA CAR T cells as a potential treatment for not only MM but also some lymphomas.

Targeting prostate cancer: Prostate-specific membrane antigen based diagnosis and therapy.

PubMed

Wüstemann, Till; Haberkorn, Uwe; Babich, John; Mier, Walter

2018-05-17

The high incidence rates of prostate cancer (PCa) raise demand for improved therapeutic strategies. Prostate tumors specifically express the prostate-specific membrane antigen (PSMA), a membrane-bound protease. As PSMA is highly overexpressed on malignant prostate tumor cells and as its expression rate correlates with the aggressiveness of the disease, this tumor-associated biomarker provides the possibility to develop new strategies for diagnostics and therapy of PCa. Major advances have been made in PSMA targeting, ranging from immunotherapeutic approaches to therapeutic small molecules. This review elaborates the diversity of PSMA targeting agents while focusing on the radioactively labeled tracers for diagnosis and endoradiotherapy. A variety of radionuclides have been shown to either enable precise diagnosis or efficiently treat the tumor with minimal effects to nontargeted organs. Most small molecules with affinity for PSMA are based on either a phosphonate or a urea-based binding motif. Based on these pharmacophores, major effort has been made to identify modifications to achieve ideal pharmacokinetics while retaining the specific targeting of the PSMA binding pocket. Several tracers have now shown excellent clinical usability in particular for molecular imaging and therapy as proven by the efficiency of theranostic approaches in current studies. The archetypal expression profile of PSMA may be exploited for the treatment with alpha emitters to break radioresistance and thus to bring the power of systemic therapy to higher levels. © 2018 Wiley Periodicals, Inc.

P‐TEFb goes viral

PubMed Central

Zaborowska, Justyna; Isa, Nur F.

2015-01-01

Positive transcription elongation factor b (P‐TEFb), which comprises cyclin‐dependent kinase 9 (CDK9) kinase and cyclin T subunits, is an essential kinase complex in human cells. Phosphorylation of the negative elongation factors by P‐TEFb is required for productive elongation of transcription of protein‐coding genes by RNA polymerase II (pol II). In addition, P‐TEFb‐mediated phosphorylation of the carboxyl‐terminal domain (CTD) of the largest subunit of pol II mediates the recruitment of transcription and RNA processing factors during the transcription cycle. CDK9 also phosphorylates p53, a tumor suppressor that plays a central role in cellular responses to a range of stress factors. Many viral factors affect transcription by recruiting or modulating the activity of CDK9. In this review, we will focus on how the function of CDK9 is regulated by viral gene products. The central role of CDK9 in viral life cycles suggests that drugs targeting the interaction between viral products and P‐TEFb could be effective anti‐viral agents. PMID:27398404

Tracking serum antibody response to viral antigens with arrayed imaging reflectometry

NASA Astrophysics Data System (ADS)

Mace, Charles R.; Rose, Robert C.; Miller, Benjamin L.

2009-02-01

Arrayed Imaging Reflectometry, or "AIR", is a new label-free technique for detecting proteins that relies on bindinginduced changes in the response of an antireflective coating on the surface of a silicon ship. Because the technique provides high sensitivity, excellent dynamic range, and readily integrates with standard silicon wafer processing technology, it is an exceptionally attractive platform on which to build systems for detecting proteins in complex solutions. In our early research, we used AIR chips bearing secreted receptor proteins from enteropathogenic E. coli to develop sensors for this pathogen. Recently, we have been exploring an alternative strategy: Rather than detecting the pathogen directly, can one immobilize antigens from a pathogen, and employ AIR to detect antibody responses to those antigens? Such a strategy would provide enhanced sensitivity for pathogen detection (as the immune system essentially amplifies the "signal" caused by the presence of an organism to which it responds), and would also potentially prove useful in the process of vaccine development. We describe herein preliminary results in the application of such a strategy to the detection of antibodies to human papillomavirus (HPV).

Controlled and targeted release of antigens by intelligent shell for improving applicability of oral vaccines.

PubMed

Zhang, Lei; Zeng, Zhanzhuang; Hu, Chaohua; Bellis, Susan L; Yang, Wendi; Su, Yintao; Zhang, Xinyan; Wu, Yunkun

2016-01-01

Conventional oral vaccines with simple architecture face barriers with regard to stimulating effective immunity. Here we describe oral vaccines with an intelligent phase-transitional shielding layer, poly[(methyl methacrylate)-co-(methyl acrylate)-co-(methacrylic acid)]-poly(D,L-lactide-co-glycolide) (PMMMA-PLGA), which can protect antigens in the gastro-intestinal tract and achieve targeted vaccination in the large intestine. With the surface immunogenic protein (SIP) from group B Streptococcus (GBS) entrapped as the antigen, oral administration with PMMMA-PLGA (PTRBL)/Trx-SIP nanoparticles stimulated robust immunity in tilapia, an animal with a relatively simple immune system. The vaccine succeeded in protecting against Streptococcus agalactiae, a pathogen of worldwide importance that threatens human health and is transmitted in water with infected fish. After oral vaccination with PTRBL/Trx-SIP, tilapia produced enhanced levels of SIP specific antibodies and displayed durability of immune protection. 100% of the vaccinated tilapia were protected from GBS infection, whereas the control groups without vaccines or vaccinated with Trx-SIP only exhibited respective infection rates of 100% or >60% within the initial 5 months after primary vaccination. Experiments in vivo demonstrated that the recombinant antigen Trx-SIP labeled with FITC was localized in colon, spleen and kidney, which are critical sites for mounting an immune response. Our results revealed that, rather than the size of the nanoparticles, it is more likely that the negative charge repulsion produced by ionization of the carboxyl groups in PMMMA shielded the nanoparticles from uptake by small intestinal epithelial cells. This system resolves challenges arising from gastrointestinal damage to antigens, and more importantly, offers a new approach applicable for oral vaccination. Copyright © 2015 Elsevier Ltd. All rights reserved.

miR-888: A Novel Cancer-Testis Antigen that Targets the Progesterone Receptor in Endometrial Cancer12

PubMed Central

Hovey, Adriann M.; Devor, Eric J.; Breheny, Patrick J.; Mott, Sarah L.; Dai, Donghai; Thiel, Kristina W.; Leslie, Kimberly K.

2015-01-01

Cancer-testis (CT) antigens are a large family of genes that are selectively expressed in human testis germ cells, overexpressed in a variety of tumors and predominantly located on the X chromosome. To date, all known CT antigens are protein-coding genes. Here, we identify miR-888 as the first miRNA with features characteristic of a CT antigen. In a panel of 21 normal human tissues, miR-888 expression was high in testes and minimal or absent in all other examined tissues. In situ hybridization localized miR-888 expression specifically to the early stages of sperm development within the testes. Using The Cancer Genome Atlas database, we discovered that miR-888 was predominately expressed in endometrial tumors, with a significant association to high-grade tumors and increased percent invasion. In a separate panel of endometrial tumor specimens, we validated overexpression of miR-888 by real-time polymerase chain reaction. In addition, miR-888 expression was highest in endometrial carcinosarcoma, a rare and aggressive type of endometrial tumor. Moreover, we identified the progesterone receptor (PR), a potent endometrial tumor suppressor, as a direct target of miR-888. These data define miR-888 as the first miRNA CT antigen and a potential mediator of an aggressive endometrial tumor phenotype through down-regulation of PR. PMID:25926074

Vaccine to Control the Viral Infection of Fish.

DOEpatents

Leong, JoAnn Ching

1994-10-11

Subunit vaccines and their use for immunizing fish against infection by viruses are disclosed. In particular, plasmid pG8 is constructed by joining, with the plasmid pUC8, DNA which encodes the glycoprotein of infectious hematopoietic necrosis virus (IHNV). E. coli cells are transformed by pG8, whereby pure viral antigen is produced to provide a vaccine for the control of IHNV in fish. 10 figs.

In vitro generation of viral-antigen dependent cytotoxic T-cells from ginbuna crucian carp, Carassius auratus langsdorfii

DOE Office of Scientific and Technical Information (OSTI.GOV)

Somamoto, Tomonori, E-mail: somamoto@agr.kyushu-u.ac.j; Okamoto, Nobuaki; Nakanishi, Teruyuki

2009-06-20

Little is known about antigen-specific T-cell responses to viruses in teleosts due to a lack of a suitable experimental system using inbred or clonal animals. In the present study we have successfully induced an in vitro generation of virus-specific cytotoxic T-cells (CTLs) from isogeneic ginbuna crucian carp. Responder cells (primarily lymphocytes) from crucian carp haematopoietic necrosis virus (CHNV)-infected fish were capable of proliferating after stimulation in vitro with CHNV-infected syngeneic stimulator cells (primarily lymphocytes and macrophages). The effector cells collected 8 and 12 days after the in vitro stimulation efficiently lysed CHNV-infected syngeneic cells, but not CHNV-infected allogeneic cells ormore » different virus (EVA)-infected syngeneic cells. Furthermore, in situ hybridization analysis showed that some effector cells binding to a CHNV-infected target were TCRbeta or CD8alpha positive. These results provide evidence that the teleost effector cells generated in vitro correspond to virus-specific CTL and they recognize virus-infected target cells in a similar manner of mammalian counterparts.« less

Structural flexibility of a conserved antigenic region in hepatitis C virus glycoprotein E2 recognized by broadly neutralizing antibodies.

PubMed

Meola, Annalisa; Tarr, Alexander W; England, Patrick; Meredith, Luke W; McClure, C Patrick; Foung, Steven K H; McKeating, Jane A; Ball, Jonathan K; Rey, Felix A; Krey, Thomas

2015-02-01

Neutralizing antibodies (NAbs) targeting glycoprotein E2 are important for the control of hepatitis C virus (HCV) infection. One conserved antigenic site (amino acids 412 to 423) is disordered in the reported E2 structure, but a synthetic peptide mimicking this site forms a β-hairpin in complex with three independent NAbs. Our structure of the same peptide in complex with NAb 3/11 demonstrates a strikingly different extended conformation. We also show that residues 412 to 423 are essential for virus entry but not for E2 folding. Together with the neutralizing capacity of the 3/11 Fab fragment, this indicates an unexpected structural flexibility within this epitope. NAbs 3/11 and AP33 (recognizing the extended and β-hairpin conformations, respectively) display similar neutralizing activities despite converse binding kinetics. Our results suggest that HCV utilizes conformational flexibility as an immune evasion strategy, contributing to the limited immunogenicity of this epitope in patients, similar to the conformational flexibility described for other enveloped and nonenveloped viruses. Approximately 180 million people worldwide are infected with hepatitis C virus (HCV), and neutralizing antibodies play an important role in controlling the replication of this major human pathogen. We show here that one of the most conserved antigenic sites within the major glycoprotein E2 (amino acids 412 to 423), which is disordered in the recently reported crystal structure of an E2 core fragment, can adopt different conformations in the context of the infectious virus particle. Recombinant Fab fragments recognizing different conformations of this antigenic site have similar neutralization activities in spite of converse kinetic binding parameters. Of note, an antibody response targeting this antigenic region is less frequent than those targeting other more immunogenic regions in E2. Our results suggest that the observed conformational flexibility in this conserved antigenic

Viral Immunotherapy to Eradicate Subclinical Brain Metastases

DTIC Science & Technology

2014-05-01

host innate and adaptive immune cells in metastases and normal tissues i. Months 6-21 ii. Basse Brain tissue with D2F2/E2 tumors from animals...and viral antigens which could activate memory T- cells in the draining lymphoid organs. Time course studies showed that virus infection produced a 2.6... lymphoid tissues. III. ACTIVATED NK CELLS LOCALIZE EFFICIENTLY AT TUMOR SITES The densities of NK cells found in well-established tumors in most

Antigen clasping by two antigen-binding sites of an exceptionally specific antibody for histone methylation

DOE PAGES

Hattori, Takamitsu; Lai, Darson; Dementieva, Irina S.; ...

2016-02-09

Antibodies have a well-established modular architecture wherein the antigen-binding site residing in the antigen-binding fragment (Fab or Fv) is an autonomous and complete unit for antigen recognition. Here, we describe antibodies departing from this paradigm. We developed recombinant antibodies to trimethylated lysine residues on histone H3, important epigenetic marks and challenging targets for molecular recognition. Quantitative characterization demonstrated their exquisite specificity and high affinity, and they performed well in common epigenetics applications. Surprisingly, crystal structures and biophysical analyses revealed that two antigen-binding sites of these antibodies form a head-to-head dimer and cooperatively recognize the antigen in the dimer interface. Thismore » “antigen clasping” produced an expansive interface where trimethylated Lys bound to an unusually extensive aromatic cage in one Fab and the histone N terminus to a pocket in the other, thereby rationalizing the high specificity. A long-neck antibody format with a long linker between the antigen-binding module and the Fc region facilitated antigen clasping and achieved both high specificity and high potency. Antigen clasping substantially expands the paradigm of antibody–antigen recognition and suggests a strategy for developing extremely specific antibodies.« less

Antigen clasping by two antigen-binding sites of an exceptionally specific antibody for histone methylation

DOE Office of Scientific and Technical Information (OSTI.GOV)

Hattori, Takamitsu; Lai, Darson; Dementieva, Irina S.

Antibodies have a well-established modular architecture wherein the antigen-binding site residing in the antigen-binding fragment (Fab or Fv) is an autonomous and complete unit for antigen recognition. Here, we describe antibodies departing from this paradigm. We developed recombinant antibodies to trimethylated lysine residues on histone H3, important epigenetic marks and challenging targets for molecular recognition. Quantitative characterization demonstrated their exquisite specificity and high affinity, and they performed well in common epigenetics applications. Surprisingly, crystal structures and biophysical analyses revealed that two antigen-binding sites of these antibodies form a head-to-head dimer and cooperatively recognize the antigen in the dimer interface. Thismore » “antigen clasping” produced an expansive interface where trimethylated Lys bound to an unusually extensive aromatic cage in one Fab and the histone N terminus to a pocket in the other, thereby rationalizing the high specificity. A long-neck antibody format with a long linker between the antigen-binding module and the Fc region facilitated antigen clasping and achieved both high specificity and high potency. Antigen clasping substantially expands the paradigm of antibody–antigen recognition and suggests a strategy for developing extremely specific antibodies.« less

Antigenic structure of soluble herpes simplex virus (HSV) glycoprotein D correlates with inhibition of HSV infection.

PubMed Central

Nicola, A V; Peng, C; Lou, H; Cohen, G H; Eisenberg, R J

1997-01-01

Soluble forms of herpes simplex virus (HSV) glycoprotein D (gD) block viral penetration. Likewise, most HSV strains are sensitive to gD-mediated interference by cells expressing gD. The mechanism of both forms of gD-mediated inhibition is thought to be at the receptor level. We analyzed the ability of different forms of soluble, truncated gD (gDt) to inhibit infection by different strains of HSV-1 and HSV-2. Strains that were resistant to gD-mediated interference were also resistant to inhibition by gDt, thereby suggesting a link between these two phenomena. Virion gD was the major viral determinant for resistance to inhibition by gDt. An insertion-deletion mutant, gD-1(delta 290-299t), had an enhanced inhibitory activity against most strains tested. The structure and function of gDt proteins derived from the inhibition-resistant viruses rid1 and ANG were analyzed. gD-1(ridlt) and gD-1(ANGt) had a potent inhibitory effect on plaque formation by wild-type strains of HSV but, surprisingly, little or no effect on their parental strains. As measured by quantitative enzyme-linked immunosorbent assay with a diverse panel of monoclonal antibodies, the antigenic structures of gD-1(rid1t) and gD-1(ANGt) were divergent from that of the wild type yet were similar to each other and to that of gD-1 (delta 290-299t). Thus, three different forms of gD have common antigenic changes that correlate with enhanced inhibitory activity against HSV. We conclude that inhibition of HSV infectivity by soluble gD is influenced by the antigenic conformation of the blocking gDt as well as the form of gD in the target virus. PMID:9060653

Synergistic co-targeting of prostate-specific membrane antigen and androgen receptor in prostate cancer.

PubMed

Murga, Jose D; Moorji, Sameer M; Han, Amy Q; Magargal, Wells W; DiPippo, Vincent A; Olson, William C

2015-02-15

Antibody-drug conjugates (ADCs) are an emerging class of cancer therapies that have demonstrated favorable activity both as single agents and as components of combination regimens. Phase 2 testing of an ADC targeting prostate-specific membrane antigen (PSMA) in advanced prostate cancer has shown antitumor activity. The present study examined PSMA ADC used in combination with potent antiandrogens (enzalutamide and abiraterone) and other compounds. Antiproliferative activity and expression of PSMA, prostate-specific antigen and androgen receptor were evaluated in the prostate cancer cell lines LNCaP and C4-2. Cells were tested for susceptibility to antiandrogens or other inhibitors, used alone and in combination with PSMA ADC. Potential drug synergy or antagonism was evaluated using the Bliss independence method. Enzalutamide and abiraterone demonstrated robust, statistically significant synergy when combined with PSMA ADC. Largely additive activity was observed between the antiandrogens and the individual components of the ADC (free drug and unmodified antibody). Rapamycin also synergized with PSMA ADC in certain settings. Synergy was linked in part to upregulation of PSMA expression. In androgen-dependent LNCaP cells, enzalutamide and abiraterone each inhibited proliferation, upregulated PSMA expression, and synergized with PSMA ADC. In androgen-independent C4-2 cells, enzalutamide and abiraterone showed no measurable antiproliferative activity on their own but increased PSMA expression and synergized with PSMA ADC nonetheless. PSMA expression increased progressively over 3 weeks with enzalutamide and returned to baseline levels 1 week after enzalutamide removal. The findings support exploration of clinical treatment regimens that combine potent antiandrogens and PSMA-targeted therapies for prostate cancer. © 2014 Wiley Periodicals, Inc.

[Microbiological diagnosis of viral respiratory infections].

PubMed

Eiros, José M; Ortiz de Lejarazu, Raúl; Tenorio, Alberto; Casas, Inmaculada; Pozo, Francisco; Ruiz, Guillermo; Pérez-Breña, Pilar

2009-03-01

Acute respiratory infection is the most common disease occurring over a person's lifetime, with etiological variations determined mainly by age, environmental circumstances, the healthcare setting, and the underlying pathology. More than 200 different viruses distributed in six viral families have been implicated in the pathogenesis of respiratory tract infection. These facts are generating an increasing diagnostic demand that should be incorporated into the healthcare setting without delay. To meet this demand, the Spanish Society of Infectious Diseases and Clinical Microbiology has updated its Standard Procedure for the microbiological diagnosis of viral respiratory infection. This document contains an update primarily of infections caused by influenza viruses, and secondarily, infections due to other conventional and emerging respiratory viruses. In all cases, the methods for direct virological diagnosis (cell culture, and detection of antigens and nucleic acid) are reviewed, with special reference to techniques for molecular detection and genetic characterization.

Adenovirus Delivered Short Hairpin RNA Targeting a Conserved Site in the 5′ Non-Translated Region Inhibits All Four Serotypes of Dengue Viruses

PubMed Central

Korrapati, Anil Babu; Swaminathan, Gokul; Singh, Aarti; Khanna, Navin; Swaminathan, Sathyamangalam

2012-01-01

Background Dengue is a mosquito-borne viral disease caused by four closely related serotypes of Dengue viruses (DENVs). This disease whose symptoms range from mild fever to potentially fatal haemorrhagic fever and hypovolemic shock, threatens nearly half the global population. There is neither a preventive vaccine nor an effective antiviral therapy against dengue disease. The difference between severe and mild disease appears to be dependent on the viral load. Early diagnosis may enable timely therapeutic intervention to blunt disease severity by reducing the viral load. Harnessing the therapeutic potential of RNA interference (RNAi) to attenuate DENV replication may offer one approach to dengue therapy. Methodology/Principal Findings We screened the non-translated regions (NTRs) of the RNA genomes of representative members of the four DENV serotypes for putative siRNA targets mapping to known transcription/translation regulatory elements. We identified a target site in the 5′ NTR that maps to the 5′ upstream AUG region, a highly conserved cis-acting element essential for viral replication. We used a replication-defective human adenovirus type 5 (AdV5) vector to deliver a short-hairpin RNA (shRNA) targeting this site into cells. We show that this shRNA matures to the cognate siRNA and is able to inhibit effectively antigen secretion, viral RNA replication and infectious virus production by all four DENV serotypes. Conclusion/Significance The data demonstrate the feasibility of using AdV5-mediated delivery of shRNAs targeting conserved sites in the viral genome to achieve inhibition of all four DENV serotypes. This paves the way towards exploration of RNAi as a possible therapeutic strategy to curtail DENV infection. PMID:22848770

Modularly Constructed Synthetic Granzyme B Molecule Enables Interrogation of Intracellular Proteases for Targeted Cytotoxicity.

PubMed

Ho, Patrick; Ede, Christopher; Chen, Yvonne Y

2017-08-18

Targeted therapies promise to increase the safety and efficacy of treatments against diseases ranging from cancer to viral infections. However, the vast majority of targeted therapeutics relies on the recognition of extracellular biomarkers, which are rarely restricted to diseased cells and are thus prone to severe and sometimes-fatal off-target toxicities. In contrast, intracellular antigens present a diverse yet underutilized repertoire of disease markers. Here, we report a protein-based therapeutic platform-termed Cytoplasmic Oncoprotein VErifier and Response Trigger (COVERT)-which enables the interrogation of intracellular proteases to trigger targeted cytotoxicity. COVERT molecules consist of the cytotoxic protein granzyme B (GrB) fused to an inhibitory N-terminal peptide, which can be removed by researcher-specified proteases to activate GrB function. We demonstrate that fusion of a small ubiquitin-like modifier 1 (SUMO1) protein to GrB yields a SUMO-GrB molecule that is specifically activated by the cancer-associated sentrin-specific protease 1 (SENP1). SUMO-GrB selectively triggers apoptotic phenotypes in HEK293T cells that overexpress SENP1, and it is highly sensitive to different SENP1 levels across cell lines. We further demonstrate the rational design of additional COVERT molecules responsive to enterokinase (EK) and tobacco etch virus protease (TEVp), highlighting the COVERT platform's modularity and adaptability to diverse protease targets. As an initial step toward engineering COVERT-T cells for adoptive T-cell therapy, we verified that primary human T cells can express, package, traffic, and deliver engineered GrB molecules in response to antigen stimulation. Our findings set the foundation for future intracellular-antigen-responsive therapeutics that can complement surface-targeted therapies.

Multiplex CRISPR/Cas9 system impairs HCMV replication by excising an essential viral gene.

PubMed

Gergen, Janina; Coulon, Flora; Creneguy, Alison; Elain-Duret, Nathan; Gutierrez, Alejandra; Pinkenburg, Olaf; Verhoeyen, Els; Anegon, Ignacio; Nguyen, Tuan Huy; Halary, Franck Albert; Haspot, Fabienne

2018-01-01

Anti-HCMV treatments used in immunosuppressed patients reduce viral replication, but resistant viral strains can emerge. Moreover, these drugs do not target latently infected cells. We designed two anti-viral CRISPR/Cas9 strategies to target the UL122/123 gene, a key regulator of lytic replication and reactivation from latency. The singleplex strategy contains one gRNA to target the start codon. The multiplex strategy contains three gRNAs to excise the complete UL122/123 gene. Primary fibroblasts and U-251 MG cells were transduced with lentiviral vectors encoding Cas9 and one or three gRNAs. Both strategies induced mutations in the target gene and a concomitant reduction of immediate early (IE) protein expression in primary fibroblasts. Further detailed analysis in U-251 MG cells showed that the singleplex strategy induced 50% of indels in the viral genome, leading to a reduction in IE protein expression. The multiplex strategy excised the IE gene in 90% of all viral genomes and thus led to the inhibition of IE protein expression. Consequently, viral genome replication and late protein expression were reduced by 90%. Finally, the production of new viral particles was nearly abrogated. In conclusion, the multiplex anti-UL122/123 CRISPR/Cas9 system can target the viral genome efficiently enough to significantly prevent viral replication.

Antigenic properties of the envelope of influenza virus rendered soluble by surfactant-solvent systems

PubMed Central

Larin, N. M.; Gallimore, P. H.

1971-01-01

Dissociating chemical treatments employing surfactant-solvent systems were applied to purified influenza A and B viruses to obtain viral preparations possessing a significantly higher or lower haemagglutinating activity than the intact virus. All preparations, whether with high or low haemagglutinating activity, with the exception of envelope protein solubilized by Triton X-100, were significantly lacking in the ability to excite the formation of haemagglutination-inhibiting and virus-neutralizing antibodies in inoculated ferrets. In contrast to other treatments, Triton X-100 treatment of virus significantly enhanced the antigenicity of viral protein as judged by virus neutralization and haemagglutination inhibition tests. Yet the haemagglutinating activity of the envelope protein solubilized with Triton X-100 was about 1% that of the intact virus. Results suggest that the correlation assumed to exist between the haemagglutinating activity of influenza virus and its ability to excite the formation of humoral antibodies is coincidental. Another important point is that the specific antigenicity of viral protein may be lost or enhanced owing to effects, other than solubilization, by surface-active agents. PMID:5291750

Use of thermodynamic coupling between antibody-antigen binding and phospholipid acyl chain phase transition energetics to predict immunoliposome targeting affinity.

PubMed

Klegerman, Melvin E; Zou, Yuejiao; Golunski, Eva; Peng, Tao; Huang, Shao-Ling; McPherson, David D

2014-09-01

Thermodynamic analysis of ligand-target binding has been a useful tool for dissecting the nature of the binding mechanism and, therefore, potentially can provide valuable information regarding the utility of targeted formulations. Based on a consistent coupling of antibody-antigen binding and gel-liquid crystal transition energetics observed for antibody-phosphatidylethanolamine (Ab-PE) conjugates, we hypothesized that the thermodynamic parameters and the affinity for antigen of the Ab-PE conjugates could be effectively predicted once the corresponding information for the unconjugated antibody is determined. This hypothesis has now been tested in nine different antibody-targeted echogenic liposome (ELIP) preparations, where antibody is conjugated to dipalmitoylphosphatidylethanolamine (DPPE) head groups through a thioether linkage. Predictions were satisfactory (affinity not significantly different from the population of values found) in five cases (55.6%), but the affinity of the unconjugated antibody was not significantly different from the population of values found in six cases (66.7%), indicating that the affinities of the conjugated antibody tended not to deviate appreciably from those of the free antibody. While knowledge of the affinities of free antibodies may be sufficient to judge their suitability as targeting agents, thermodynamic analysis may still provide valuable information regarding their usefulness for specific applications.

Targeting tumor antigens to secreted membrane vesicles in vivo induces efficient antitumor immune responses.

PubMed

Zeelenberg, Ingrid S; Ostrowski, Matias; Krumeich, Sophie; Bobrie, Angélique; Jancic, Carolina; Boissonnas, Alexandre; Delcayre, Alain; Le Pecq, Jean-Bernard; Combadière, Béhazine; Amigorena, Sebastian; Théry, Clotilde

2008-02-15

Expression of non-self antigens by tumors can induce activation of T cells in vivo, although this activation can lead to either immunity or tolerance. CD8+ T-cell activation can be direct (if the tumor expresses MHC class I molecules) or indirect (after the capture and cross-presentation of tumor antigens by dendritic cells). The modes of tumor antigen capture by dendritic cells in vivo remain unclear. Here we examine the immunogenicity of the same model antigen secreted by live tumors either in association with membrane vesicles (exosomes) or as a soluble protein. We have artificially addressed the antigen to secreted vesicles by coupling it to the factor VIII-like C1C2 domain of milk fat globule epidermal growth factor-factor VIII (MFG-E8)/lactadherin. We show that murine fibrosarcoma tumor cells that secrete vesicle-bound antigen grow slower than tumors that secrete soluble antigen in immunocompetent, but not in immunodeficient, host mice. This growth difference is due to the induction of a more potent antigen-specific antitumor immune response in vivo by the vesicle-bound than by the soluble antigen. Finally, in vivo secretion of the vesicle-bound antigen either by tumors or by vaccination with naked DNA protects against soluble antigen-secreting tumors. We conclude that the mode of secretion can determine the immunogenicity of tumor antigens and that manipulation of the mode of antigen secretion may be used to optimize antitumor vaccination protocols.

Engineering antigens for in situ erythrocyte binding induces T-cell deletion.

PubMed

Kontos, Stephan; Kourtis, Iraklis C; Dane, Karen Y; Hubbell, Jeffrey A

2013-01-02

Antigens derived from apoptotic cell debris can drive clonal T-cell deletion or anergy, and antigens chemically coupled ex vivo to apoptotic cell surfaces have been shown correspondingly to induce tolerance on infusion. Reasoning that a large number of erythrocytes become apoptotic (eryptotic) and are cleared each day, we engineered two different antigen constructs to target the antigen to erythrocyte cell surfaces after i.v. injection, one using a conjugate with an erythrocyte-binding peptide and another using a fusion with an antibody fragment, both targeting the erythrocyte-specific cell surface marker glycophorin A. Here, we show that erythrocyte-binding antigen is collected much more efficiently than free antigen by splenic and hepatic immune cell populations and hepatocytes, and that it induces antigen-specific deletional responses in CD4(+) and CD8(+) T cells. We further validated T-cell deletion driven by erythrocyte-binding antigens using a transgenic islet β cell-reactive CD4(+) T-cell adoptive transfer model of autoimmune type 1 diabetes: Treatment with the peptide antigen fused to an erythrocyte-binding antibody fragment completely prevented diabetes onset induced by the activated, autoreactive CD4(+) T cells. Thus, we report a translatable modular biomolecular approach with which to engineer antigens for targeted binding to erythrocyte cell surfaces to induce antigen-specific CD4(+) and CD8(+) T-cell deletion toward exogenous antigens and autoantigens.

Targeting endosomal acidification by chloroquine analogs as a promising strategy for the treatment of emerging viral diseases.

PubMed

Al-Bari, Md Abdul Alim

2017-02-01

Emerging viruses such as HIV, dengue, influenza A, SARS coronavirus, Ebola, and other viruses pose a significant threat to human health. Majority of these viruses are responsible for the outbreaks of pathogenic lethal infections. To date, there are no effective therapeutic strategies available for the prophylaxis and treatment of these infections. Chloroquine analogs have been used for decades as the primary and most successful drugs against malaria. Concomitant with the emergence of chloroquine-resistant Plasmodium strains and a subsequent decrease in the use as antimalarial drugs, other applications of the analogs have been investigated. Since the analogs have interesting biochemical properties, these drugs are found to be effective against a wide variety of viral infections. As antiviral action, the analogs have been shown to inhibit acidification of endosome during the events of replication and infection. Moreover, immunomodulatory effects of analogs have been beneficial to patients with severe inflammatory complications of several viral diseases. Interestingly, one of the successful targeting strategies is the inhibition of HIV replication by the analogs in vitro which are being tested in several clinical trials. This review focuses on the potentialities of chloroquine analogs for the treatment of endosomal low pH dependent emerging viral diseases.

PA28γ is a novel corepressor of HTLV-1 replication and controls viral latency

PubMed Central

Ko, Nga Ling; Taylor, John M.; Bellon, Marcia; Bai, Xue Tao; Shevtsov, Sergey P.; Dundr, Miroslav

2013-01-01

The establishment of a latent reservoir by human tumor viruses is a vital step in initiating cellular transformation and represents a major shortcoming to current therapeutic strategies and the ability to eradicate virus-infected cells. Human T-cell leukemia virus type 1 (HTLV-1) establishes a lifelong infection and is linked to adult T-cell leukemia lymphoma (ATLL). Here, we demonstrate that HTLV-1 p30 recruits the cellular proteasome activator PA28γ onto the viral tax/rex mRNA to prevent its nuclear export and suppress virus replication. Interaction of p30 with a PA28γ retaining fully functional proteasome activity is required for p30's ability to repress HTLV-1. Consistently, HTLV-1 molecular clones replicate better and produce more virus particles in PA28γ-deficient cells. These results define a unique and novel role for the cellular factor PA28γ in the control of nuclear RNA trafficking and HTLV-1–induced latency. Importantly, knockdown of PA28γ expression in ATLL cells latently infected with HTLV-1 reactivates expression of viral tax/rex RNA and the Tax protein. Because Tax is the most immunogenic viral antigen and triggers strong CTL responses, our results suggest that PA28γ-targeted therapy may reactivate virus expression from latently infected cells and allow their eradication from the host. PMID:23104922

Measles virus induces persistent infection by autoregulation of viral replication.

PubMed

Doi, Tomomitsu; Kwon, Hyun-Jeong; Honda, Tomoyuki; Sato, Hiroki; Yoneda, Misako; Kai, Chieko

2016-11-24

Natural infection with measles virus (MV) establishes lifelong immunity. Persistent infection with MV is likely involved in this phenomenon, as non-replicating protein antigens never induce such long-term immunity. Although MV establishes stable persistent infection in vitro and possibly in vivo, the mechanism by which this occurs is largely unknown. Here, we demonstrate that MV changes the infection mode from lytic to non-lytic and evades the innate immune response to establish persistent infection without viral genome mutation. We found that, in the persistent phase, the viral RNA level declined with the termination of interferon production and cell death. Our analysis of viral protein dynamics shows that during the establishment of persistent infection, the nucleoprotein level was sustained while the phosphoprotein and large protein levels declined. The ectopic expression of nucleoprotein suppressed viral replication, indicating that viral replication is self-regulated by nucleoprotein accumulation during persistent infection. The persistently infected cells were able to produce interferon in response to poly I:C stimulation, suggesting that MV does not interfere with host interferon responses in persistent infection. Our results may provide mechanistic insight into the persistent infection of this cytopathic RNA virus that induces lifelong immunity.

Antigen-inducing ability of herpesvirus papio in human and baboon lymphoma lines, compared to Epstein-Barr virus.

PubMed

Klein, G; Falk, L; Falk, K

1978-01-01

Herpesvirus papio(HVP)-carrying baboon lymphoblastoid lines do not express a nuclear antigen like the Epstein-Barr virus(EBV)-determined nuclear antigen (EBNA), as judged by in situ anticomplement fluorescence staining, although the carry multiple viral genomes and, in the case of producerlines, early antigen (EA) and viral capsid antigen (VCA) that cross-react with the corresponding human EBV-determined antigens. To test whether the lack of in situ nuclear antigen expression is a property innate to the baboon virus or the baboon cell, nonproducer HVP-carrying baboon lymphoid cells of the 26 CB-1 line were superinfected with two human EBV strains. B95-8-derived EBV induced brilliant EBNA staining, proving that the baboon lymphoid cell was competent to synthesize EBNA. In the mirror experiment, HVP derived from the 9B or the 18C baboon line was added to the EBV-carrying Raji line, the EBV-negative Ramos and BJAB lines and the HVP-carrying nonproducer 26 CB-1 line, respectively. HVP induced EA and VCA in Raji, and EA in BJAB and 26 CB-1. EBNA was not induced in any of the three EBNA-negative lines, BJAB, Ramos and 26 CB-1. It is concluded that the lack of in situ nuclear staining in HVP-carrying baboon lines is a HVP-associated property and is not due to any innate inability of the baboon lymphoid cell to synthesize an antigen of the EBNA type.

EBNA1: Oncogenic Activity, Immune Evasion and Biochemical Functions Provide Targets for Novel Therapeutic Strategies against Epstein-Barr Virus- Associated Cancers

PubMed Central

Wilson, Joanna B.; Manet, Evelyne; Fahraeus, Robin

2018-01-01

The presence of the Epstein-Barr virus (EBV)-encoded nuclear antigen-1 (EBNA1) protein in all EBV-carrying tumours constitutes a marker that distinguishes the virus-associated cancer cells from normal cells and thereby offers opportunities for targeted therapeutic intervention. EBNA1 is essential for viral genome maintenance and also for controlling viral gene expression and without EBNA1, the virus cannot persist. EBNA1 itself has been linked to cell transformation but the underlying mechanism of its oncogenic activity has been unclear. However, recent data are starting to shed light on its growth-promoting pathways, suggesting that targeting EBNA1 can have a direct growth suppressing effect. In order to carry out its tasks, EBNA1 interacts with cellular factors and these interactions are potential therapeutic targets, where the aim would be to cripple the virus and thereby rid the tumour cells of any oncogenic activity related to the virus. Another strategy to target EBNA1 is to interfere with its expression. Controlling the rate of EBNA1 synthesis is critical for the virus to maintain a sufficient level to support viral functions, while at the same time, restricting expression is equally important to prevent the immune system from detecting and destroying EBNA1-positive cells. To achieve this balance EBNA1 has evolved a unique repeat sequence of glycines and alanines that controls its own rate of mRNA translation. As the underlying molecular mechanisms for how this repeat suppresses its own rate of synthesis in cis are starting to be better understood, new therapeutic strategies are emerging that aim to modulate the translation of the EBNA1 mRNA. If translation is induced, it could increase the amount of EBNA1-derived antigenic peptides that are presented to the major histocompatibility (MHC) class I pathway and thus, make EBV-carrying cancers better targets for the immune system. If translation is further suppressed, this would provide another means to cripple

Human Adenovirus Core Protein V Is Targeted by the Host SUMOylation Machinery To Limit Essential Viral Functions.

PubMed

Freudenberger, Nora; Meyer, Tina; Groitl, Peter; Dobner, Thomas; Schreiner, Sabrina

2018-02-15

Human adenoviruses (HAdV) are nonenveloped viruses containing a linear, double-stranded DNA genome surrounded by an icosahedral capsid. To allow proper viral replication, the genome is imported through the nuclear pore complex associated with viral core proteins. Until now, the role of these incoming virion proteins during the early phase of infection was poorly understood. The core protein V is speculated to bridge the core and the surrounding capsid. It binds the genome in a sequence-independent manner and localizes in the nucleus of infected cells, accumulating at nucleoli. Here, we show that protein V contains conserved SUMO conjugation motifs (SCMs). Mutation of these consensus motifs resulted in reduced SUMOylation of the protein; thus, protein V represents a novel target of the host SUMOylation machinery. To understand the role of protein V SUMO posttranslational modification during productive HAdV infection, we generated a replication-competent HAdV with SCM mutations within the protein V coding sequence. Phenotypic analyses revealed that these SCM mutations are beneficial for adenoviral replication. Blocking protein V SUMOylation at specific sites shifts the onset of viral DNA replication to earlier time points during infection and promotes viral gene expression. Simultaneously, the altered kinetics within the viral life cycle are accompanied by more efficient proteasomal degradation of host determinants and increased virus progeny production than that observed during wild-type infection. Taken together, our studies show that protein V SUMOylation reduces virus growth; hence, protein V SUMOylation represents an important novel aspect of the host antiviral strategy to limit virus replication and thereby points to potential intervention strategies. IMPORTANCE Many decades of research have revealed that HAdV structural proteins promote viral entry and mainly physical stability of the viral genome in the capsid. Our work over the last years showed that this

Characterization of O-antigen delivered by Generalized Modules for Membrane Antigens (GMMA) vaccine candidates against nontyphoidal Salmonella.

PubMed

De Benedetto, G; Alfini, R; Cescutti, P; Caboni, M; Lanzilao, L; Necchi, F; Saul, A; MacLennan, C A; Rondini, S; Micoli, F

2017-01-11

Invasive nontyphoidal Salmonella disease (iNTS) is a leading cause of death and morbidity in Africa. The most common pathogens are Salmonella enterica serovars Typhimurium and Enteritidis. The O-antigen portion of their lipopolysaccharide is a target of protective immunity and vaccines targeting O-antigen are currently in development. Here we investigate the use of Generalized Modules for Membrane Antigens (GMMA) as delivery system for S. Typhimurium and S. Enteritidis O-antigen. Gram-negative bacteria naturally shed outer membrane in a blebbing process. By deletion of the tolR gene, the level of shedding was greatly enhanced. Further genetic modifications were introduced into the GMMA-producing strains in order to reduce reactogenicity, by detoxifying the lipid A moiety of lipopolysaccharide. We found that genetic mutations can impact on expression of O-antigen chains. All S. Enteritidis GMMA characterized had an O-antigen to protein w/w ratio higher than 0.6, while the ratio was 0.7 for S. Typhimurium ΔtolR GMMA, but decreased to less than 0.1 when further mutations for lipid A detoxification were introduced. Changes were also observed in O-antigen chain length and level and/or position of O-acetylation. When tested in mice, the GMMA induced high levels of anti-O-antigen-specific IgG functional antibodies, despite variation in density and O-antigen structural modifications. In conclusion, simplicity of manufacturing process and low costs of production, coupled with encouraging immunogenicity data, make GMMA an attractive strategy to further investigate for the development of a vaccine against iNTS. Copyright © 2016. Published by Elsevier Ltd.

Specific Nongluten Proteins of Wheat Are Novel Target Antigens in Celiac Disease Humoral Response

PubMed Central

2014-01-01

While the antigenic specificity and pathogenic relevance of immunologic reactivity to gluten in celiac disease have been extensively researched, the immune response to nongluten proteins of wheat has not been characterized. We aimed to investigate the level and molecular specificity of antibody response to wheat nongluten proteins in celiac disease. Serum samples from patients and controls were screened for IgG and IgA antibody reactivity to a nongluten protein extract from the wheat cultivar Triticum aestivum Butte 86. Antibodies were further analyzed for reactivity to specific nongluten proteins by two-dimensional gel electrophoresis and immunoblotting. Immunoreactive molecules were identified by tandem mass spectrometry. Compared with healthy controls, patients exhibited significantly higher levels of antibody reactivity to nongluten proteins. The main immunoreactive nongluten antibody target proteins were identified as serpins, purinins, α-amylase/protease inhibitors, globulins, and farinins. Assessment of reactivity toward purified recombinant proteins further confirmed the presence of antibody response to specific antigens. The results demonstrate that, in addition to the well-recognized immune reaction to gluten, celiac disease is associated with a robust humoral response directed at a specific subset of the nongluten proteins of wheat. PMID:25329597

Targeting Stereotyped B Cell Receptors from Chronic Lymphocytic Leukemia Patients with Synthetic Antigen Surrogates.

PubMed

Sarkar, Mohosin; Liu, Yun; Qi, Junpeng; Peng, Haiyong; Morimoto, Jumpei; Rader, Christoph; Chiorazzi, Nicholas; Kodadek, Thomas

2016-04-01

Chronic lymphocytic leukemia (CLL) is a disease in which a single B-cell clone proliferates relentlessly in peripheral lymphoid organs, bone marrow, and blood. DNA sequencing experiments have shown that about 30% of CLL patients have stereotyped antigen-specific B-cell receptors (BCRs) with a high level of sequence homology in the variable domains of the heavy and light chains. These include many of the most aggressive cases that haveIGHV-unmutated BCRs whose sequences have not diverged significantly from the germ line. This suggests a personalized therapy strategy in which a toxin or immune effector function is delivered selectively to the pathogenic B-cells but not to healthy B-cells. To execute this strategy, serum-stable, drug-like compounds able to target the antigen-binding sites of most or all patients in a stereotyped subset are required. We demonstrate here the feasibility of this approach with the discovery of selective, high affinity ligands for CLL BCRs of the aggressive, stereotyped subset 7P that cross-react with the BCRs of several CLL patients in subset 7p, but not with BCRs from patients outside this subset. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

Recent Advances in Non-viral Vectors for Gene Delivery

PubMed Central

Guo, Xia; Huang, Leaf

2011-01-01

CONSPECTUS Non-viral vectors, typically based on cationic lipids or polymers, are preferred due to safety concerns with viral vectors. So far, non-viral vectors can proficiently transfect cells in culture, but obtaining efficient nanomedicines is far from evident. To overcome the hurdles associated with non-viral vectors is significant for improving delivery efficiency and therapeutic effect of nucleic acid. The drawbacks include the strong interaction of cationic delivery vehicles with blood components, uptake by the reticuloendothelial system (RES), toxicity, targeting ability of the carriers to the cells of interest, and so on. PEGylation is the predominant method used to reduce the binding of plasma proteins with non-viral vectors and minimize the clearance by RES after intravenous administration. The nanoparticles that are not rapidly cleared from the circulation accumulate in the tumors due to the enhanced permeability and retention effect, and the targeting ligands attached to the distal end of the PEGylated components allow binding to the receptors on the target cell surface. Neutral or anionic liposomes have been also developed for systemic delivery of nucleic acids in experimental animal model. Designing and synthesizing novel cationic lipids and polymers, and binding nucleic acid with peptides, targeting ligands, polymers, or environmentally sensitive moieties also attract many attentions for resolving the problems encountered by non-viral vectors. The application of inorganic nanoparticles in nucleic acid delivery is an emerging field, too. Recently, different classes of non-viral vectors appear to be converging and the features of different classes of non-viral vectors could be combined in one strategy. More hurdles associated with efficient nucleic acid delivery therefore might be expected to be overcome. In this account, we will focus on these novel non-viral vectors, which are classified into multifunctional hybrid nucleic acid vectors, novel

[The lysate and recombinant antigens in ELISA-test-systems for diagnostic of herpes simplex].

PubMed

Ganova, L A; Kovtoniuk, G V; Korshun, L N; Kiseleva, E K; Tereshchenko, M I; Vudmaska, M I; Moĭsa, L N; Shevchuk, V A; Spivak, N Ia

2014-08-01

The lysate and recombinant antigens of various production included informula of ELISA-test-systems were analyzed. The ELISA-test-systems are used for detection of IgG to Herpes simplex virus type I and II. For testing the panel of serums PTH 201 (BBI Inc.) were used. The samples of this panel contain antibodies to Herpes simplex virus type I and II in mixed titers. The 69 serums of donors were used too (17 samples had IgG to Herpes simplex virus type I, 23 samples to Herpes simplex virus type II and 29 samples had no antibodies to Herpes simplex virus). The diagnostic capacity of mixture of recombinant antigens gG1 Herpes simplex virus type I and gG2 Herpes simplex virus type II (The research-and-production complex "DiaprofMed") was comparable with mixture of lysate antigen Herpes simplex virus type I and II (Membrane) EIE Antigen ("Virion Ltd."). In the test-systems for differentiation of IgG to Herpes simplex virus type I the recombinant antigen gG1 Herpes simplex virus type I proved to be comparable with commercial analogue Herpes simplex virus-1 gG1M ("Viral Therapeutics Inc."'). At the same time, capacity to detect IgG to Herpes simplex virus type II in recombinant protein gG2 Herpes simplex virus type II is significantly higher than in its analogue Herpes simplex virus-2 gG2c ("Viral Therapeutics Inc.").

Immune Response to Recombinant Adenovirus in Humans: Capsid Components from Viral Input Are Targets for Vector-Specific Cytotoxic T Lymphocytes

PubMed Central

Molinier-Frenkel, Valérie; Gahery-Segard, Hanne; Mehtali, Majid; Le Boulaire, Christophe; Ribault, Sébastien; Boulanger, Pierre; Tursz, Thomas; Guillet, Jean-Gérard; Farace, Françoise

2000-01-01

We previously demonstrated that a single injection of 109 PFU of recombinant adenovirus into patients induces strong vector-specific immune responses (H. Gahéry-Ségard, V. Molinier-Frenkel, C. Le Boulaire, P. Saulnier, P. Opolon, R. Lengagne, E. Gautier, A. Le Cesne, L. Zitvogel, A. Venet, C. Schatz, M. Courtney, T. Le Chevalier, T. Tursz, J.-G. Guillet, and F. Farace, J. Clin. Investig. 100:2218–2226, 1997). In the present study we analyzed the mechanism of vector recognition by cytotoxic T lymphocytes (CTL). CD8+ CTL lines were derived from two patients and maintained in long-term cultures. Target cell infections with E1-deleted and E1-plus E2-deleted adenoviruses, as well as transcription-blocking experiments with actinomycin D, revealed that host T-cell recognition did not require viral gene transcription. Target cells treated with brefeldin A were not lysed, indicating that viral input protein-derived peptides are associated with HLA class I molecules. Using recombinant capsid component-loaded targets, we observed that the three major proteins could be recognized. These results raise the question of the use of multideleted adenoviruses for gene therapy in the quest to diminish antivector CTL responses. PMID:10906225

Overcoming immune tolerance in chronic hepatitis B by therapeutic vaccination.

PubMed

Dembek, Claudia; Protzer, Ulrike; Roggendorf, Michael

2018-05-08

The currently used nucleoside analogs (i.e. entecavir and tenofovir) with high barrier-to-resistance efficiently suppress viral replication, limit inflammation and reduce the sequelae of chronic hepatitis B, but cannot cure the disease and thus have to be applied long-term. Therapeutic vaccination as an approach to cure chronic hepatitis B has shown promising pre-clinical results, nevertheless the proof of its efficacy in clinical trials is still missing. This may be partially due to suboptimal vaccine design. A main obstacle in chronic hepatitis B, however, is the high load of viral antigens expressed and secreted, which has been proposed to cause antigen-specific immune tolerance. Reduction of the viral antigen load is therefore considered a key factor for success of immune-based therapies. Although nucleoside analogs do not reduce viral antigen expression, new antiviral strategies are becoming available. Targeting viral translation by siRNA or targeting release of HBsAg from infected hepatocytes by nucleic acid polymers both reduce the antigen load. They may be considered as pre-treatment for therapeutic vaccination to increase the potential to elicit an HBV-specific immune response able to control and cure chronic HBV infection. Copyright © 2018 Elsevier B.V. All rights reserved.

Anti-CD22–chimeric antigen receptors targeting B-cell precursor acute lymphoblastic leukemia

PubMed Central

Haso, Waleed; Lee, Daniel W.; Shah, Nirali N.; Stetler-Stevenson, Maryalice; Yuan, Constance M.; Pastan, Ira H.; Dimitrov, Dimiter S.; Morgan, Richard A.; FitzGerald, David J.; Barrett, David M.; Wayne, Alan S.; Mackall, Crystal L.

2013-01-01

Immune targeting of B-cell malignancies using chimeric antigen receptors (CARs) is a promising new approach, but critical factors impacting CAR efficacy remain unclear. To test the suitability of targeting CD22 on precursor B-cell acute lymphoblastic leukemia (BCP-ALL), lymphoblasts from 111 patients with BCP-ALL were assayed for CD22 expression and all were found to be CD22-positive, with median CD22 expression levels of 3500 sites/cell. Three distinct binding domains targeting CD22 were fused to various TCR signaling domains ± an IgG heavy chain constant domain (CH2CH3) to create a series of vector constructs suitable to delineate optimal CAR configuration. CARs derived from the m971 anti-CD22 mAb, which targets a proximal CD22 epitope demonstrated superior antileukemic activity compared with those incorporating other binding domains, and addition of a 4-1BB signaling domain to CD28.CD3ζ constructs diminished potency, whereas increasing affinity of the anti-CD22 binding motif, and extending the CD22 binding domain away from the membrane via CH2CH3 had no effect. We conclude that second-generation m971 mAb-derived anti-CD22 CARs are promising novel therapeutics that should be tested in BCP-ALL. PMID:23243285

Higher-order looping and nuclear organization of antigen receptor loci facilitate targeted RAG cleavage and regulated rearrangement in recombination centers

PubMed Central

Chaumeil, Julie; Micsinai, Mariann; Ntziachristos, Panagiotis; Deriano, Ludovic; Wang, Joy M-H; Ji, Yanhong; Nora, Elphege P.; Rodesch, Matthew J.; Jeddeloh, Jeffrey A.; Aifantis, Iannis; Kluger, Yuval; Schatz, David G.; Skok, Jane A.

2013-01-01

SUMMARY V(D)J recombination is essential for generating a diverse array of B and T cell receptors that can recognize and combat foreign antigen. As with any recombination event, tight control is essential to prevent the occurrence of genetic anomalies that drive cellular transformation. One important aspect of regulation is directed targeting of the RAG recombinase. Indeed, RAG accumulates at the 3’ end of individual antigen receptor loci poised for rearrangement, however, it is not known whether focal binding is involved in regulating cleavage, and what mechanisms lead to enrichment of RAG in this region. Here we show that mono-allelic looping out of the 3’ end of Tcra, coupled with transcription and increased chromatin/nuclear accessibility, are linked to focal RAG binding and ATM-mediated regulated mono-allelic cleavage on looped out 3’ regions. Our data identify higher order loop formation as a key determinant of directed RAG targeting and the maintenance of genome stability. PMID:23416051

Structural characterization of viral epitopes recognized by broadly cross-reactive antibodies.

PubMed

Lee, Peter S; Wilson, Ian A

2015-01-01

Influenza hemagglutinin (HA) is the major surface glycoprotein on influenza viruses and mediates viral attachment and subsequent fusion with host cells. The HA is the major target of the immune response, but due to its high level of variability, as evidenced by substantial antigenic diversity, it had been historically considered to elicit only a narrow, strain-specific antibody response. However, a recent explosion in the discovery of broadly neutralizing antibodies (bnAbs) to influenza virus has identified two major supersites of vulnerability on the HA through structural characterization of HA-antibody complexes. These commonly targeted epitopes are involved with receptor binding as well as the fusion machinery and, hence, are functionally conserved and less prone to mutation. These bnAbs can neutralize viruses by blocking infection or the spread of infection by preventing progeny release. Structural analyses of these bnAbs show they exhibit striking similarities and trends in recognition of the HA and use recurring recognition motifs, despite substantial differences in their germline genes. This information can be utilized in design of novel therapeutics as well as in immunogens for improved vaccines with greater breadth and efficacy.

Structure, Receptor Binding, and Antigenicity of Influenza Virus Hemagglutinins from the 1957 H2N2 Pandemic

DOE Office of Scientific and Technical Information (OSTI.GOV)

Xu, Rui; McBride, Ryan; Paulson, James C.

2010-03-04

The hemagglutinin (HA) envelope protein of influenza viruses mediates essential viral functions, including receptor binding and membrane fusion, and is the major viral antigen for antibody neutralization. The 1957 H2N2 subtype (Asian flu) was one of the three great influenza pandemics of the last century and caused 1 million deaths globally from 1957 to 1968. Three crystal structures of 1957 H2 HAs have been determined at 1.60 to 1.75 {angstrom} resolutions to investigate the structural basis for their antigenicity and evolution from avian to human binding specificity that contributed to its introduction into the human population. These structures, which representmore » the highest resolutions yet recorded for a complete ectodomain of a glycosylated viral surface antigen, along with the results of glycan microarray binding analysis, suggest that a hydrophobicity switch at residue 226 and elongation of receptor-binding sites were both critical for avian H2 HA to acquire human receptor specificity. H2 influenza viruses continue to circulate in birds and pigs and, therefore, remain a substantial threat for transmission to humans. The H2 HA structure also reveals a highly conserved epitope that could be harnessed in the design of a broader and more universal influenza A virus vaccine.« less

Cidofovir inhibits polyomavirus BK replication in human renal tubular cells downstream of viral early gene expression.

PubMed

Bernhoff, E; Gutteberg, T J; Sandvik, K; Hirsch, H H; Rinaldo, C H

2008-07-01

The human polyomavirus BK (BKV) causes nephropathy and hemorrhagic cystitis in kidney and bone marrow transplant patients, respectively. The anti-viral cidofovir (CDV) has been used in small case series but the effects on BKV replication are unclear, since polyomaviruses do not encode viral DNA polymerases. We investigated the effects of CDV on BKV(Dunlop) replication in primary human renal proximal tubule epithelial cells (RPTECs). CDV inhibited the generation of viral progeny in a dose-dependent manner yielding a 90% reduction at 40 microg/mL. Early steps such as receptor binding and entry seemed unaffected. Initial large T-antigen transcription and expression were also unaffected, but subsequent intra-cellular BKV DNA replication was reduced by >90%. Late viral mRNA and corresponding protein levels were also 90% reduced. In uninfected RPTECs, CDV 40 microg/mL reduced cellular DNA replication and metabolic activity by 7% and 11% in BrdU and WST-1 assays, respectively. BKV infection increased DNA replication to 142% and metabolic activity to 116%, respectively, which were reduced by CDV 40 microg/mL to levels of uninfected untreated RPTECs. Our results show that CDV inhibits BKV DNA replication downstream of large T-antigen expression and involves significant host cell toxicity. This should be considered in current treatment and drug development.

Antigen Loss Variants: Catching Hold of Escaping Foes.

PubMed

Vyas, Maulik; Müller, Rolf; Pogge von Strandmann, Elke

2017-01-01

Since mid-1990s, the field of cancer immunotherapy has seen steady growth and selected immunotherapies are now a routine and preferred therapeutic option of certain malignancies. Both active and passive cancer immunotherapies exploit the fact that tumor cells express specific antigens on the cell surface, thereby mounting an immune response specifically against malignant cells. It is well established that cancer cells typically lose surface antigens following natural or therapy-induced selective pressure and these antigen-loss variants are often the population that causes therapy-resistant relapse. CD19 and CD20 antigen loss in acute lymphocytic leukemia and chronic lymphocytic leukemia, respectively, and lineage switching in leukemia associated with mixed lineage leukemia (MLL) gene rearrangements are well-documented evidences in this regard. Although increasing number of novel immunotherapies are being developed, majority of these do not address the control of antigen loss variants. Here, we review the occurrence of antigen loss variants in leukemia and discuss the therapeutic strategies to tackle the same. We also present an approach of dual-targeting immunoligand effectively retargeting NK cells against antigen loss variants in MLL-associated leukemia. Novel immunotherapies simultaneously targeting more than one tumor antigen certainly hold promise to completely eradicate tumor and prevent therapy-resistant relapses.

Prophage-mediated defense against viral attack and viral counter-defense

PubMed Central

Dedrick, Rebekah M.; Jacobs-Sera, Deborah; Guerrero Bustamante, Carlos A.; Garlena, Rebecca A.; Mavrich, Travis N.; Pope, Welkin H.; Reyes, Juan C Cervantes; Russell, Daniel A.; Adair, Tamarah; Alvey, Richard; Bonilla, J. Alfred; Bricker, Jerald S.; Brown, Bryony R.; Byrnes, Deanna; Cresawn, Steven G.; Davis, William B.; Dickson, Leon A.; Edgington, Nicholas P.; Findley, Ann M.; Golebiewska, Urszula; Grose, Julianne H.; Hayes, Cory F.; Hughes, Lee E.; Hutchison, Keith W.; Isern, Sharon; Johnson, Allison A.; Kenna, Margaret A.; Klyczek, Karen K.; Mageeney, Catherine M.; Michael, Scott F.; Molloy, Sally D.; Montgomery, Matthew T.; Neitzel, James; Page, Shallee T.; Pizzorno, Marie C.; Poxleitner, Marianne K.; Rinehart, Claire A.; Robinson, Courtney J.; Rubin, Michael R.; Teyim, Joseph N.; Vazquez, Edwin; Ware, Vassie C.; Washington, Jacqueline; Hatfull, Graham F.

2017-01-01

Temperate phages are common and prophages are abundant residents of sequenced bacterial genomes. Mycobacteriophages are viruses infecting mycobacterial hosts including Mycobacterium tuberculosis and Mycobacterium smegmatis, encompass substantial genetic diversity, and are commonly temperate. Characterization of ten Cluster N temperate mycobacteriophages reveals at least five distinct prophage-expressed viral defense systems that interfere with infection of lytic and temperate phages that are either closely-related (homotypic defense) or unrelated (heterotypic defense). Target specificity is unpredictable, ranging from a single target phage to one-third of those tested. The defense systems include a single-subunit restriction system, a heterotypic exclusion system, and a predicted (p)ppGpp synthetase, which blocks lytic phage growth, promotes bacterial survival, and enables efficient lysogeny. The predicted (p)ppGpp synthetase coded by the Phrann prophage defends against phage Tweety infection, but Tweety codes for a tetrapeptide repeat protein, gp54, that acts as a highly effective counter-defense system. Prophage-mediated viral defense offers an efficient mechanism for bacterial success in host-virus dynamics, and counter-defense promotes phage co-evolution. PMID:28067906

A filamentous bacteriophage targeted to carcinoembryonic antigen induces tumor regression in mouse models of colorectal cancer.

PubMed

Murgas, Paola; Bustamante, Nicolás; Araya, Nicole; Cruz-Gómez, Sebastián; Durán, Eduardo; Gaete, Diana; Oyarce, César; López, Ernesto; Herrada, Andrés Alonso; Ferreira, Nicolás; Pieringer, Hans; Lladser, Alvaro

2018-02-01

Colorectal cancer is a deadly disease, which is frequently diagnosed at advanced stages, where conventional treatments are no longer effective. Cancer immunotherapy has emerged as a new form to treat different malignancies by turning-on the immune system against tumors. However, tumors are able to evade antitumor immune responses by promoting an immunosuppressive microenvironment. Single-stranded DNA containing M13 bacteriophages are highly immunogenic and can be specifically targeted to the surface of tumor cells to trigger inflammation and infiltration of activated innate immune cells, overcoming tumor-associated immunosuppression and promoting antitumor immunity. Carcinoembryonic antigen (CEA) is highly expressed in colorectal cancers and has been shown to promote several malignant features of colorectal cancer cells. In this work, we targeted M13 bacteriophage to CEA, a tumor-associated antigen over-expressed in a high proportion of colorectal cancers but largely absent in normal cells. The CEA-targeted M13 bacteriophage was shown to specifically bind to purified CEA and CEA-expressing tumor cells in vitro. Both intratumoral and systemic administration of CEA-specific bacteriophages significantly reduced tumor growth of mouse models of colorectal cancer, as compared to PBS and control bacteriophage administration. CEA-specific bacteriophages promoted tumor infiltration of neutrophils and macrophages, as well as maturation dendritic cells in tumor-draining lymph nodes, suggesting that antitumor T-cell responses were elicited. Finally, we demonstrated that tumor protection provided by CEA-specific bacteriophage particles is mediated by CD8 + T cells, as depletion of circulating CD8 + T cells completely abrogated antitumor protection. In summary, we demonstrated that CEA-specific M13 bacteriophages represent a potential immunotherapy against colorectal cancer.

Inhibition of Nipah virus infection in vivo: targeting an early stage of paramyxovirus fusion activation during viral entry.

PubMed

Porotto, Matteo; Rockx, Barry; Yokoyama, Christine C; Talekar, Aparna; Devito, Ilaria; Palermo, Laura M; Liu, Jie; Cortese, Riccardo; Lu, Min; Feldmann, Heinz; Pessi, Antonello; Moscona, Anne

2010-10-28

In the paramyxovirus cell entry process, receptor binding triggers conformational changes in the fusion protein (F) leading to viral and cellular membrane fusion. Peptides derived from C-terminal heptad repeat (HRC) regions in F have been shown to inhibit fusion by preventing formation of the fusogenic six-helix bundle. We recently showed that the addition of a cholesterol group to HRC peptides active against Nipah virus targets these peptides to the membrane where fusion occurs, dramatically increasing their antiviral effect. In this work, we report that unlike the untagged HRC peptides, which bind to the postulated extended intermediate state bridging the viral and cell membranes, the cholesterol tagged HRC-derived peptides interact with F before the fusion peptide inserts into the target cell membrane, thus capturing an earlier stage in the F-activation process. Furthermore, we show that cholesterol tagging renders these peptides active in vivo: the cholesterol-tagged peptides cross the blood brain barrier, and effectively prevent and treat in an established animal model what would otherwise be fatal Nipah virus encephalitis. The in vivo efficacy of cholesterol-tagged peptides, and in particular their ability to penetrate the CNS, suggests that they are promising candidates for the prevention or therapy of infection by Nipah and other lethal paramyxoviruses.

T Cells that Recognize HPV Protein Can Target Virus-Infected Cells | Center for Cancer Research

Cancer.gov

Adoptive T-cell transfer (ACT) is a promising form of cancer immunotherapy. Treating patients with T cells isolated from a tumor and subsequently expanded in the lab can cause the complete regression of some melanomas and cervical cancers, but the treatment is currently restricted to a few cancer types. An approach that may be applied to a wider array of cancers involves modifying peripheral blood T cells with chimeric antigen receptors or T-cell receptors (TCR) that target specific tumor antigens. Unfortunately, epithelial cancers, which are the vast majority of cancers diagnosed, have proven difficult to treat this way because most identified antigens are shared with healthy tissues and targeting them leads to toxic side effects. However, cancers caused by persistent human papillomavirus (HPV) infection, including cervical, head and neck, anal, vaginal, vulvar, and penile cancers, may be particularly amenable to the latter form of ACT since the E6 and E7 viral proteins are essential for cancer formation but are not produced in normal tissues. To test this idea, Christian Hinrichs, M.D., and his colleagues examined tumor infiltrating lymphocytes (TILs) from a patient who experienced a prolonged disease-free period after her second surgical removal of metastatic anal cancer in the hopes of identifying a TCR against one of the HPV oncoproteins.

New Potent Membrane-Targeting Antibacterial Peptides from Viral Capsid Proteins

PubMed Central

Dias, Susana A.; Freire, João M.; Pérez-Peinado, Clara; Domingues, Marco M.; Gaspar, Diana; Vale, Nuno; Gomes, Paula; Andreu, David; Henriques, Sónia T.; Castanho, Miguel A. R. B.; Veiga, Ana S.

2017-01-01

The increasing prevalence of multidrug-resistant bacteria urges the development of new antibacterial agents. With a broad spectrum activity, antimicrobial peptides have been considered potential antibacterial drug leads. Using bioinformatic tools we have previously shown that viral structural proteins are a rich source for new bioactive peptide sequences, namely antimicrobial and cell-penetrating peptides. Here, we test the efficacy and mechanism of action of the most promising peptides among those previously identified against both Gram-positive and Gram-negative bacteria. Two cell-penetrating peptides, vCPP 0769 and vCPP 2319, have high antibacterial activity against Staphylococcus aureus, MRSA, Escherichia coli, and Pseudomonas aeruginosa, being thus multifunctional. The antibacterial mechanism of action of the two most active viral protein-derived peptides, vAMP 059 and vCPP 2319, was studied in detail. Both peptides act on both Gram-positive S. aureus and Gram-negative P. aeruginosa, with bacterial cell death occurring within minutes. Also, these peptides cause bacterial membrane permeabilization and damage of the bacterial envelope of P. aeruginosa cells. Overall, the results show that structural viral proteins are an abundant source for membrane-active peptides sequences with strong antibacterial properties. PMID:28522994

Effects of transmission-blocking vaccines simultaneously targeting pre- and post-fertilization antigens in the rodent malaria parasite Plasmodium yoelii.

PubMed

Zheng, Li; Pang, Wei; Qi, Zanmei; Luo, Enjie; Cui, Liwang; Cao, Yaming

2016-08-08

Transmission-blocking vaccine (TBV) is a promising strategy for interrupting the malaria transmission cycle. Current TBV candidates include both pre- and post-fertilization antigens expressed during sexual development of the malaria parasites. We tested whether a TBV design combining two sexual-stage antigens has better transmission-blocking activity. Using the rodent malaria model Plasmodium yoelii, we pursued a DNA vaccination strategy with genes encoding the gametocyte antigen Pys48/45 and the major ookinete surface protein Pys25. Immunization of mice with DNA constructs expression either Pys48/45 or Pys25 elicited strong antibody responses, which specifically recognized a ~45 and ~25 kDa protein from gametocyte and ookinete lysates, respectively. Immune sera from mice immunized with DNA constructs expressing Pys48/45 and Pys25 individually and in combination displayed evident transmission-blocking activity in in vitro ookinete culture and direct mosquito feeding experiments. With both assays, the Pys25 sera had higher transmission-blocking activity than the Pys48/45 sera. Intriguingly, compared with the immunization with the individual DNA vaccines, immunization with both DNA constructs produced lower antibody responses against individual antigens. The resultant immune sera from the composite vaccination had significantly lower transmission-blocking activity than those from Pys25 DNA immunization group, albeit the activity was substantially higher than that from the Pys48 DNA vaccination group. This result suggested that vaccination with the two DNA constructs did not achieve a synergistic effect, but rather caused interference in inducing antigen-specific antibody responses. This result has important implications for future design of composite vaccines targeting different sexual antigens.

Human Monoclonal Islet Cell Antibodies From a Patient with Insulin- Dependent Diabetes Mellitus Reveal Glutamate Decarboxylase as the Target Antigen

NASA Astrophysics Data System (ADS)

Richter, Wiltrud; Endl, Josef; Eiermann, Thomas H.; Brandt, Michael; Kientsch-Engel, Rosemarie; Thivolet, Charles; Jungfer, Herbert; Scherbaum, Werner A.

1992-09-01

The autoimmune phenomena associated with destruction of the β cell in pancreatic islets and development of type 1 (insulin-dependent) diabetes mellitus (IDDM) include circulating islet cell antibodies. We have immortalized peripheral blood lymphocytes from prediabetic individuals and patients with newly diagnosed IDDM by Epstein-Barr virus transformation. IgG-positive cells were selected by anti-human IgG-coupled magnetic beads and expanded in cell culture. Supernatants were screened for cytoplasmic islet cell antibodies using the conventional indirect immunofluorescence test on cryostat sections of human pancreas. Six islet cell-specific B-cell lines, originating from a patient with newly diagnosed IDDM, could be stabilized on a monoclonal level. All six monoclonal islet cell antibodies (MICA 1-6) were of the IgG class. None of the MICA reacted with human thyroid, adrenal gland, anterior pituitary, liver, lung, stomach, and intestine tissues but all six reacted with pancreatic islets of different mammalian species and, in addition, with neurons of rat cerebellar cortex. MICA 1-6 were shown to recognize four distinct antigenic epitopes in islets. Islet cell antibody-positive diabetic sera but not normal human sera blocked the binding of the monoclonal antibodies to their target epitopes. Immunoprecipitation of 35S-labeled human islet cell extracts revealed that a protein of identical size to the enzyme glutamate decarboxylase (EC 4.1.1.15) was a target of all MICA. Furthermore, antigen immunotrapped by the MICA from brain homogenates showed glutamate decarboxylase enzyme activity. MICA 1-6 therefore reveal glutamate decarboxylase as the predominant target antigen of cytoplasmic islet cell autoantibodies in a patient with newly diagnosed IDDM.

Non-viral nucleic acid containing nanoparticles as cancer therapeutics.

PubMed

Kozielski, Kristen L; Rui, Yuan; Green, Jordan J

2016-10-01

The delivery of nucleic acids such as DNA and short interfering RNA (siRNA) is promising for the treatment of many diseases, including cancer, by enabling novel biological mechanisms of action. Non-viral nanoparticles are a promising class of nucleic acid carriers that can be designed to be safer and more versatile than traditional viral vectors. In this review, recent advances in the intracellular delivery of DNA and siRNA are described with a focus on non-viral nanoparticle-based delivery methods. Material properties that have enabled successful delivery are discussed as well as applications that have directly been applied to cancer therapy. Strategies to co-deliver different nucleic acids are highlighted, as are novel targets for nucleic acid co-delivery. The treatment of complex genetically-based diseases such as cancer can be enabled by safe and effective intracellular delivery of multiple nucleic acids. Non-viral nanoparticles can be fabricated to deliver multiple nucleic acids to the same cell simultaneously to prevent tumor cells from easily compensating for the knockdown or overexpression of one genetic target. The continued innovation of new therapeutic modalities and non-viral nanotechnologies to provide target-specific and personalized forms of gene therapy hold promise for genetic medicine to treat diseases like cancer in the clinic.

Effect of temperature post viral vector inoculation on the amount of hemagglutinin transiently expressed in Nicotiana benthamiana leaves.

PubMed

Matsuda, Ryo; Abe, Tatsuki; Fujiuchi, Naomichi; Matoba, Nobuyuki; Fujiwara, Kazuhiro

2017-09-01

Transient gene expression in whole plants by using viral vectors is promising as a rapid, mass production system for biopharmaceutical proteins. Recent studies have indicated that plant growth conditions such as air temperature markedly influence the accumulation levels of target proteins. Here, we investigated time course of the amount of recombinant hemagglutinin (HA), a vaccine antigen of influenza virus, in leaves of Nicotiana benthamiana plants grown at 20°C or 25°C post viral vector inoculation. The HA content per unit of leaf biomass increased and decreased from 4 to 6 days post inoculation at 20°C and 25°C, respectively, irrespective of the subcellular localization of HA. The overall HA contents were higher when HA was targeted to the endoplasmic reticulum (ER) rather than the apoplast. Necrosis of leaf tissues was specifically observed in plants inoculated with the ER-targeting vector and grown at 25°C. With the ER-targeting vector, the maximum HA contents at 20°C and 25°C were recorded at 6 and 4 days post inoculation, respectively, and were comparable to each other. HA contents thereafter decreased at both temperatures; the rate of reduction appeared faster at 25°C than at 20°C. From a practical point of view, our results indicate that the strategy of targeting HA to the ER, growing plants at a lower temperature of 20°C, and harvesting leaves at around a week after vector inoculation should be implemented to obtain a high HA yield stably and efficiently. Copyright © 2017 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

Prostate-specific membrane antigen-targeted liposomes specifically deliver the Zn(2+) chelator TPEN inducing oxidative stress in prostate cancer cells.

PubMed

Stuart, Christopher H; Singh, Ravi; Smith, Thomas L; D'Agostino, Ralph; Caudell, David; Balaji, K C; Gmeiner, William H

2016-05-01

To evaluate the potential use of zinc chelation for prostate cancer therapy using a new liposomal formulation of the zinc chelator, N,N,N',N'-tetrakis(2-pyridylmethyl)-ethylenediamine (TPEN). TPEN was encapsulated in nontargeted liposomes or liposomes displaying an aptamer to target prostate cancer cells overexpression prostate-specific membrane antigen. The prostate cancer selectivity and therapeutic efficacy of liposomal (targeted and nontargeted) and free TPEN were evaluated in vitro and in tumor-bearing mice. TPEN chelates zinc and results in reactive oxygen species imbalance leading to cell death. Delivery of TPEN using aptamer-targeted liposomes results in specific delivery to targeted cells. In vivo experiments show that TPEN-loaded, aptamer-targeted liposomes reduce tumor growth in a human prostate cancer xenograft model.

TIA-1 and TIAR interact with 5'-UTR of enterovirus 71 genome and facilitate viral replication.

PubMed

Wang, Xiaohui; Wang, Huanru; Li, Yixuan; Jin, Yu; Chu, Ying; Su, Airong; Wu, Zhiwei

2015-10-16

Enterovirus 71 is one of the major causative pathogens of HFMD in children. Upon infection, the viral RNA is translated in an IRES-dependent manner and requires several host factors for effective replication. Here, we found that T-cell-restricted intracellular antigen 1 (TIA-1), and TIA-1 related protein (TIAR) were translocated from nucleus to cytoplasm after EV71 infection and localized to the sites of viral replication. We found that TIA-1 and TIAR can facilitate EV71 replication by enhancing the viral genome synthesis in host cells. We demonstrated that both proteins bound to the stem-loop I of 5'-UTR of viral genome and improved the stability of viral genomic RNA. Our results suggest that TIA-1 and TIAR are two new host factors that interact with 5-UTR of EV71 genome and positively regulate viral replication. Copyright © 2015 Elsevier Inc. All rights reserved.

Viral kinetics are associated with changes in cytokines and chemokines in serum and target organs of SSM-CVB3-infected macaques.

PubMed

Han, Tiesuo; Zhao, Kui; Wu, Chenchen; Lu, Huijun; Song, Deguang; He, Wenqi; Gao, Feng

2013-02-01

To determine the relationship between viral kinetics and the expression patterns for different cytokines and chemokines in the serum and organs of coxsackievirus B3 (SSM-CVB3)-infected macaques over the course of infection. SSM-CVB3 levels in serum and organs were measured using the Spearman-Karber 50% tissue culture infectious dose (TCID(50)) method. Cytokine and chemokine levels in the serum and organs were measured by indirect-ELISA. Low viral titers were detected in the serum samples on the first day post-inoculation (p.i.) and peaked at 6 to 10 days p.i. in the serum samples from five macaques. Serum levels of IL-1β, IL-2, IL-6, IL-12p40, IL-17α, IFN-γ, TNF-α, MCP-1 and MIP-1β were detected each day and, similar to the viral titers, peaked at 6 to 10 days. IL-10 was only detected on days 10 to 14 p.i. Additionally, higher viral titers and relative viral mRNA levels were associated with higher cytokine and chemokine levels in selected tissues from infected macaques including heart, liver, spleen, lung, kidney and brain. The results indicate that patterns of cytokine and chemokine response are associated with viral kinetics in the serum and target organs of SSM-CVB3-infected macaques, suggesting that the changes in cytokines and chemokines could help further our understanding of the progress of CVB3 infections in clinical settings. Copyright © 2012 Elsevier Inc. All rights reserved.

The PP4R1 sub-unit of protein phosphatase PP4 is essential for inhibition of NF-κB by merkel polyomavirus small tumour antigen.

PubMed

Abdul-Sada, Hussein; Müller, Marietta; Mehta, Rajni; Toth, Rachel; Arthur, J Simon C; Whitehouse, Adrian; Macdonald, Andrew

2017-04-11

Merkel cell carcinoma (MCC) is a highly aggressive skin cancer with a high metastatic potential. The majority of MCC cases are caused by the Merkel cell polyomavirus (MCPyV), through expression of the virus-encoded tumour antigens. Whilst mechanisms attributing tumour antigen expression to transformation are being uncovered, little is known of the mechanisms by which MCPyV persists in the host. We previously identified the MCPyV small T antigen (tAg) as a novel inhibitor of nuclear factor kappa B (NF-kB) signalling and a modulator of the host anti-viral response. Here we demonstrate that regulation of NF-kB activation involves a previously undocumented interaction between tAg and regulatory sub-unit 1 of protein phosphatase 4 (PP4R1). Formation of a complex with PP4R1 and PP4c is required to bridge MCPyV tAg to the NEMO adaptor protein, allowing deactivation of the NF-kB pathway. Mutations in MCPyV tAg that fail to interact with components of this complex, or siRNA depletion of PP4R1, prevents tAg-mediated inhibition of NF-kB and pro-inflammatory cytokine production. Comparison of tAg binding partners from other human polyomavirus demonstrates that interactions with NEMO and PP4R1 are unique to MCPyV. Collectively, these data identify PP4R1 as a novel target for virus subversion of the host anti-viral response.

Identifying Early Target Cells of Nipah Virus Infection in Syrian Hamsters

PubMed Central

Baseler, Laura; Scott, Dana P.; Saturday, Greg; Horne, Eva; Rosenke, Rebecca; Thomas, Tina; Meade-White, Kimberly; Haddock, Elaine; Feldmann, Heinz

2016-01-01

Background Nipah virus causes respiratory and neurologic disease with case fatality rates up to 100% in individual outbreaks. End stage lesions have been described in the respiratory and nervous systems, vasculature and often lymphoid organs in fatal human cases; however, the initial target organs of Nipah virus infection have not been identified. Here, we detected the initial target tissues and cells of Nipah virus and tracked virus dissemination during the early phase of infection in Syrian hamsters inoculated with a Nipah virus isolate from Malaysia (NiV-M) or Bangladesh (NiV-B). Methodology/Principal Findings Syrian hamsters were euthanized between 4 and 48 hours post intranasal inoculation and tissues were collected and analyzed for the presence of viral RNA, viral antigen and infectious virus. Virus replication was first detected at 8 hours post inoculation (hpi). Nipah virus initially targeted type I pneumocytes, bronchiolar respiratory epithelium and alveolar macrophages in the lung and respiratory and olfactory epithelium lining the nasal turbinates. By 16 hpi, virus disseminated to epithelial cells lining the larynx and trachea. Although the pattern of viral dissemination was similar for both virus isolates, the rate of spread was slower for NiV-B. Infectious virus was not detected in the nervous system or blood and widespread vascular infection and lesions within lymphoid organs were not observed, even at 48 hpi. Conclusions/Significance Nipah virus initially targets the respiratory system. Virus replication in the brain and infection of blood vessels in non-respiratory tissues does not occur during the early phase of infection. However, virus replicates early in olfactory epithelium and may serve as the first step towards nervous system dissemination, suggesting that development of vaccines that block virus dissemination or treatments that can access the brain and spinal cord and directly inhibit virus replication may be necessary for preventing central

Identifying Early Target Cells of Nipah Virus Infection in Syrian Hamsters.

PubMed

Baseler, Laura; Scott, Dana P; Saturday, Greg; Horne, Eva; Rosenke, Rebecca; Thomas, Tina; Meade-White, Kimberly; Haddock, Elaine; Feldmann, Heinz; de Wit, Emmie

2016-11-01

Nipah virus causes respiratory and neurologic disease with case fatality rates up to 100% in individual outbreaks. End stage lesions have been described in the respiratory and nervous systems, vasculature and often lymphoid organs in fatal human cases; however, the initial target organs of Nipah virus infection have not been identified. Here, we detected the initial target tissues and cells of Nipah virus and tracked virus dissemination during the early phase of infection in Syrian hamsters inoculated with a Nipah virus isolate from Malaysia (NiV-M) or Bangladesh (NiV-B). Syrian hamsters were euthanized between 4 and 48 hours post intranasal inoculation and tissues were collected and analyzed for the presence of viral RNA, viral antigen and infectious virus. Virus replication was first detected at 8 hours post inoculation (hpi). Nipah virus initially targeted type I pneumocytes, bronchiolar respiratory epithelium and alveolar macrophages in the lung and respiratory and olfactory epithelium lining the nasal turbinates. By 16 hpi, virus disseminated to epithelial cells lining the larynx and trachea. Although the pattern of viral dissemination was similar for both virus isolates, the rate of spread was slower for NiV-B. Infectious virus was not detected in the nervous system or blood and widespread vascular infection and lesions within lymphoid organs were not observed, even at 48 hpi. Nipah virus initially targets the respiratory system. Virus replication in the brain and infection of blood vessels in non-respiratory tissues does not occur during the early phase of infection. However, virus replicates early in olfactory epithelium and may serve as the first step towards nervous system dissemination, suggesting that development of vaccines that block virus dissemination or treatments that can access the brain and spinal cord and directly inhibit virus replication may be necessary for preventing central nervous system pathology.

NEP: web server for epitope prediction based on antibody neutralization of viral strains with diverse sequences.

PubMed

Chuang, Gwo-Yu; Liou, David; Kwong, Peter D; Georgiev, Ivelin S

2014-07-01

Delineation of the antigenic site, or epitope, recognized by an antibody can provide clues about functional vulnerabilities and resistance mechanisms, and can therefore guide antibody optimization and epitope-based vaccine design. Previously, we developed an algorithm for antibody-epitope prediction based on antibody neutralization of viral strains with diverse sequences and validated the algorithm on a set of broadly neutralizing HIV-1 antibodies. Here we describe the implementation of this algorithm, NEP (Neutralization-based Epitope Prediction), as a web-based server. The users must supply as input: (i) an alignment of antigen sequences of diverse viral strains; (ii) neutralization data for the antibody of interest against the same set of antigen sequences; and (iii) (optional) a structure of the unbound antigen, for enhanced prediction accuracy. The prediction results can be downloaded or viewed interactively on the antigen structure (if supplied) from the web browser using a JSmol applet. Since neutralization experiments are typically performed as one of the first steps in the characterization of an antibody to determine its breadth and potency, the NEP server can be used to predict antibody-epitope information at no additional experimental costs. NEP can be accessed on the internet at http://exon.niaid.nih.gov/nep. Published by Oxford University Press on behalf of Nucleic Acids Research 2014. This work is written by (a) US Government employee(s) and is in the public domain in the US.

Theranostics of prostate cancer: from molecular imaging to precision molecular radiotherapy targeting the prostate specific membrane antigen.

PubMed

Kulkarni, Harshad R; Singh, Aviral; Langbein, Thomas; Schuchardt, Christiane; Mueller, Dirk; Zhang, Jingjing; Lehmann, Coline; Baum, Richard P

2018-06-01

Alterations at the molecular level are a hallmark of cancer. Prostate cancer is associated with the overexpression of prostate-specific membrane antigen (PSMA) in a majority of cases, predominantly in advanced tumors, increasing with the grade or Gleason's score. PSMA can be selectively targeted using radiolabeled PSMA ligands. These small molecules binding the PSMA can be radiolabeled with γ-emitters like 99m Tc and 111 In or positron emitters like 68 Ga and 18 F for diagnosis as well as with their theranostic pairs such as 177 Lu (β-emitter) or 225 Ac (α-emitter) for therapy. This review summarizes the theranostic role of PSMA ligands for molecular imaging and targeted molecular radiotherapy, moving towards precision oncology.

Galactofuranose antigens, a target for diagnosis of fungal infections in humans

PubMed Central

Marino, Carla; Rinflerch, Adriana; de Lederkremer, Rosa M

2017-01-01

The use of biomarkers for the detection of fungal infections is of interest to complement histopathological and culture methods. Since the production of antibodies in immunocompromised patients is scarce, detection of a specific antigen could be effective for early diagnosis. D-Galactofuranose (Galf) is the antigenic epitope in glycoconjugates of several pathogenic fungi. Since Galf is not biosynthesized by mammals, it is an attractive candidate for diagnosis of infection. A monoclonal antibody that recognizes Galf is commercialized for detection of aspergillosis. The linkage of Galf in the natural glycans and the chemical structures of the synthesized Galf-containing oligosaccharides are described in this paper. The oligosaccharides could be used for the synthesis of artificial carbohydrate-based antigens, not enough exploited for diagnosis. PMID:28883999

Galactofuranose antigens, a target for diagnosis of fungal infections in humans.

PubMed

Marino, Carla; Rinflerch, Adriana; de Lederkremer, Rosa M

2017-08-01

The use of biomarkers for the detection of fungal infections is of interest to complement histopathological and culture methods. Since the production of antibodies in immunocompromised patients is scarce, detection of a specific antigen could be effective for early diagnosis. D-Galactofuranose (Gal f ) is the antigenic epitope in glycoconjugates of several pathogenic fungi. Since Gal f is not biosynthesized by mammals, it is an attractive candidate for diagnosis of infection. A monoclonal antibody that recognizes Gal f is commercialized for detection of aspergillosis. The linkage of Gal f in the natural glycans and the chemical structures of the synthesized Gal f -containing oligosaccharides are described in this paper. The oligosaccharides could be used for the synthesis of artificial carbohydrate-based antigens, not enough exploited for diagnosis.

Prostate-Specific Membrane Antigen Targeted Therapy of Prostate Cancer Using a DUPA-Paclitaxel Conjugate.

PubMed

Lv, Qingzhi; Yang, Jincheng; Zhang, Ruoshi; Yang, Zimeng; Yang, Zhengtao; Wang, Yongjun; Xu, Youjun; He, Zhonggui

2018-05-07

Prostate cancer (PCa) is the most prevalent cancer among men in the United States and remains the second-leading cause of cancer mortality in men. Paclitaxel (PTX) is the first line chemotherapy for PCa treatment, but its therapeutic efficacy is greatly restricted by the nonspecific distribution in vivo. Prostate-specific membrane antigen (PSMA) is overexpressed on the surface of most PCa cells, and its expression level increases with cancer aggressiveness, while being present at low levels in normal cells. The high expression level of PSMA in PCa cells offers an opportunity for target delivery of nonspecific cytotoxic drugs to PCa cells, thus improving therapeutic efficacy and reducing toxicity. PSMA has high affinity for DUPA, a glutamate urea ligand. Herein, a novel DUPA-PTX conjugate is developed using DUPA as the targeting ligand to deliver PTX specifically for treatment of PSMA expressing PCa. The targeting ligand DUPA enhances the transport capability and selectivity of PTX to tumor cells via PSMA mediated endocytosis. Besides, DUPA is conjugated with PTX via a disulfide bond, which facilitates the rapid and differential drug release in tumor cells. The DUPA-PTX conjugate exhibits potent cytotoxicity in PSMA expressing cell lines and induces a complete cessation of tumor growth with no obvious toxicity. Our findings give new insight into the PSMA-targeted delivery of chemotherapeutics and provide an opportunity for the development of novel active targeting drug delivery systems for PCa therapy.

Membrane dynamics associated with viral infection.

PubMed

de Armas-Rillo, Laura; Valera, María-Soledad; Marrero-Hernández, Sara; Valenzuela-Fernández, Agustín

2016-05-01

Viral replication and spreading are fundamental events in the viral life cycle, accounting for the assembly and egression of nascent virions, events that are directly associated with viral pathogenesis in target hosts. These processes occur in cellular compartments that are modified by specialized viral proteins, causing a rearrangement of different cell membranes in infected cells and affecting the ER, mitochondria, Golgi apparatus, vesicles and endosomes, as well as processes such as autophagic membrane flux. In fact, the activation or inhibition of membrane trafficking and other related activities are fundamental to ensure the adequate replication and spreading of certain viruses. In this review, data will be presented that support the key role of membrane dynamics in the viral cycle, especially in terms of the assembly, egression and infection processes. By defining how viruses orchestrate these events it will be possible to understand how they successfully complete their route of infection, establishing viral pathogenesis and provoking disease. © 2015 The Authors Reviews in Medical Virology Published by John Wiley & Sons, Ltd.

Vaccination of cattle against bovine viral diarrhea virus.

PubMed

Newcomer, Benjamin W; Chamorro, Manuel F; Walz, Paul H

2017-07-01

Bovine viral diarrhea virus (BVDV) is responsible for significant losses to the cattle industry. Currently, modified-live viral (MLV) and inactivated viral vaccines are available against BVDV, often in combination with other viral and bacterial antigens. Inactivated and MLV vaccines provide cattle producers and veterinarians safe and efficacious options for herd immunization to limit disease associated with BVDV infection. Vaccination of young cattle against BVDV is motivated by prevention of clinical disease and limiting viral spread to susceptible animals. For reproductive-age cattle, vaccination to prevent viremia and birth of persistently infected offspring is considered more important, while also more difficult to achieve than prevention of clinical disease. Recent advances have been made in the understanding of BVDV vaccine efficacy. In terms of preventing clinical disease, current BVDV vaccines have been demonstrated to have a rapid onset of immunity and MLV vaccines can be effectively utilized in calves possessing maternal immunity. For reproductive protection, more recent studies using multivalent MLV vaccines have demonstrated consistent fetal protection rates in the range of 85-100% in experimental studies. Proper timing and administration of BVDV vaccines can be utilized to maximize vaccine efficacy to provide an important contribution to reducing risks associated with BVDV infection. With improvements in vaccine formulations and increased understanding of the protective immune response following vaccination, control of BVDV through vaccination can be enhanced. Copyright © 2017. Published by Elsevier B.V.

TanCAR: A Novel Bispecific Chimeric Antigen Receptor for Cancer Immunotherapy

PubMed Central

Grada, Zakaria; Hegde, Meenakshi; Byrd, Tiara; Shaffer, Donald R; Ghazi, Alexia; Brawley, Vita S; Corder, Amanda; Schönfeld, Kurt; Koch, Joachim; Dotti, Gianpietro; Heslop, Helen E; Gottschalk, Stephen; Wels, Winfried S; Baker, Matthew L; Ahmed, Nabil

2013-01-01

Targeted T cells are emerging as effective non-toxic therapies for cancer. Multiple elements, however, contribute to the overall pathogenesis of cancer through both distinct and redundant mechanisms. Hence, targeting multiple cancer-specific markers simultaneously could result in better therapeutic efficacy. We created a functional chimeric antigen receptor—the TanCAR, a novel artificial molecule that mediates bispecific activation and targeting of T cells. We demonstrate the feasibility of cumulative integration of structure and docking simulation data using computational tools to interrogate the design and predict the functionality of such a complex bispecific molecule. Our prototype TanCAR induced distinct T cell reactivity against each of two tumor restricted antigens, and produced synergistic enhancement of effector functions when both antigens were simultaneously encountered. Furthermore, the TanCAR preserved the cytolytic ability of T cells upon loss of one of the target molecules and better controlled established experimental tumors by recognition of both targets in an animal disease model. This proof-of-concept approach can be used to increase the specificity of effector cells for malignant versus normal target cells, to offset antigen escape or to allow for targeting the tumor and its microenvironment. PMID:23839099

Chimeric Antigen Receptor Therapy for Cancer

PubMed Central

Barrett, David M.; Singh, Nathan; Porter, David L.; Grupp, Stephan A.; June, Carl H.

2014-01-01

Improved outcomes for patients with cancer hinge on the development of new targeted therapies with acceptable short-term and long-term toxicity. Progress in basic, preclinical, and clinical arenas spanning cellular immunology, synthetic biology, and cell-processing technologies has paved the way for clinical applications of chimeric antigen receptor– based therapies. This new form of targeted immunotherapy merges the exquisite targeting specificity of monoclonal antibodies with the potent cytotoxicity and long-term persistence provided by cytotoxic T cells. Although this field is still in its infancy, clinical trials have already shown clinically significant antitumor activity in neuroblastoma, chronic lymphocytic leukemia, and B cell lymphoma, and trials targeting a variety of other adult and pediatric malignancies are under way. Ongoing work is focused on identifying optimal tumor targets and on elucidating and manipulating both cell- and host-associated factors to support expansion and persistence of the genetically engineered cells in vivo. The potential to target essentially any tumor-associated cell-surface antigen for which a monoclonal antibody can be made opens up an entirely new arena for targeted therapy of cancer. PMID:24274181

Calcium-dependent antigen binding as a novel modality for antibody recycling by endosomal antigen dissociation

PubMed Central

Hironiwa, N; Ishii, S; Kadono, S; Iwayanagi, Y; Mimoto, F; Habu, K; Igawa, T; Hattori, K

2016-01-01

The pH-dependent antigen binding antibody, termed a recycling antibody, has recently been reported as an attractive type of second-generation engineered therapeutic antibody. A recycling antibody can dissociate antigen in the acidic endosome, and thus bind to its antigen multiple times. As a consequence, a recycling antibody can neutralize large amounts of antigen in plasma. Because this approach relies on histidine residues to achieve pH-dependent antigen binding, which could limit the epitopes that can be targeted and affect the rate of antigen dissociation in the endosome, we explored an alternative approach for generating recycling antibodies. Since calcium ion concentration is known to be lower in endosome than in plasma, we hypothesized that an antibody with antigen-binding properties that are calcium-dependent could be used as recycling antibody. Here, we report a novel anti-interleukin-6 receptor (IL-6R) antibody, identified from a phage library that binds to IL-6R only in the presence of a calcium ion. Thermal dynamics and a crystal structure study revealed that the calcium ion binds to the heavy chain CDR3 region (HCDR3), which changes and possibly stabilizes the structure of HCDR3 to make it bind to antigen calcium dependently (PDB 5AZE). In vitro and in vivo studies confirmed that this calcium-dependent antigen-binding antibody can dissociate its antigen in the endosome and accelerate antigen clearance from plasma, making it a novel approach for generating recycling antibody. PMID:26496237

Transgene expression in target-defined neuron populations mediated by retrograde infection with adeno-associated viral vectors.

PubMed

Rothermel, Markus; Brunert, Daniela; Zabawa, Christine; Díaz-Quesada, Marta; Wachowiak, Matt

2013-09-18

Tools enabling the manipulation of well defined neuronal subpopulations are critical for probing complex neuronal networks. Cre recombinase (Cre) mouse driver lines in combination with the Cre-dependent expression of proteins using viral vectors--in particular, recombinant adeno-associated viral vectors (rAAVs)--have emerged as a widely used platform for achieving transgene expression in specified neural populations. However, the ability of rAAVs to further specify neuronal subsets on the basis of their anatomical connectivity has been reported as limited or inconsistent. Here, we systematically tested a variety of widely used neurotropic rAAVs for their ability to mediate retrograde gene transduction in the mouse brain. We tested pseudotyped rAAVs of several common serotypes (rAAV 2/1, 2/5, and 2/9) as well as constructs both with and without Cre-dependent expression switches. Many of the rAAVs tested--in particular, though not exclusively, Cre-dependent vectors--showed a robust capacity for retrograde infection and transgene expression. Retrograde expression was successful over distances as large as 6 mm and in multiple neuron types, including olfactory projection neurons, neocortical pyramidal cells projecting to distinct targets, and corticofugal and modulatory projection neurons. Retrograde infection using transgenes such as ChR2 allowed for optical control or optically assisted electrophysiological identification of neurons defined genetically as well as by their projection target. These results establish a widely accessible tool for achieving combinatorial specificity and stable, long-term transgene expression to isolate precisely defined neuron populations in the intact animal.

Transgene Expression in Target-Defined Neuron Populations Mediated by Retrograde Infection with Adeno-Associated Viral Vectors

PubMed Central

Rothermel, Markus; Brunert, Daniela; Zabawa, Christine; Díaz-Quesada, Marta

2013-01-01

Tools enabling the manipulation of well defined neuronal subpopulations are critical for probing complex neuronal networks. Cre recombinase (Cre) mouse driver lines in combination with the Cre-dependent expression of proteins using viral vectors—in particular, recombinant adeno-associated viral vectors (rAAVs)—have emerged as a widely used platform for achieving transgene expression in specified neural populations. However, the ability of rAAVs to further specify neuronal subsets on the basis of their anatomical connectivity has been reported as limited or inconsistent. Here, we systematically tested a variety of widely used neurotropic rAAVs for their ability to mediate retrograde gene transduction in the mouse brain. We tested pseudotyped rAAVs of several common serotypes (rAAV 2/1, 2/5, and 2/9) as well as constructs both with and without Cre-dependent expression switches. Many of the rAAVs tested—in particular, though not exclusively, Cre-dependent vectors—showed a robust capacity for retrograde infection and transgene expression. Retrograde expression was successful over distances as large as 6 mm and in multiple neuron types, including olfactory projection neurons, neocortical pyramidal cells projecting to distinct targets, and corticofugal and modulatory projection neurons. Retrograde infection using transgenes such as ChR2 allowed for optical control or optically assisted electrophysiological identification of neurons defined genetically as well as by their projection target. These results establish a widely accessible tool for achieving combinatorial specificity and stable, long-term transgene expression to isolate precisely defined neuron populations in the intact animal. PMID:24048849

Masked Chimeric Antigen Receptor for Tumor-Specific Activation.

PubMed

Han, Xiaolu; Bryson, Paul D; Zhao, Yifan; Cinay, Gunce E; Li, Si; Guo, Yunfei; Siriwon, Natnaree; Wang, Pin

2017-01-04

Adoptive cellular therapy based on chimeric antigen receptor (CAR)-engineered T (CAR-T) cells is a powerful form of cancer immunotherapy. CAR-T cells can be redirected to specifically recognize tumor-associated antigens (TAAs) and induce high levels of antitumor activity. However, they may also display "on-target off-tumor" toxicities, resulting from low-level expression of TAAs in healthy tissues. These adverse effects have raised considerable safety concerns and limited the clinical application of this otherwise promising therapeutic modality. To minimize such side effects, we have designed an epidermal growth factor receptor (EGFR)-specific masked CAR (mCAR), which consists of a masking peptide that blocks the antigen-binding site and a protease-sensitive linker. Proteases commonly active in the tumor microenvironment can cleave the linker and disengage the masking peptide, thereby enabling CAR-T cells to recognize target antigens only at the tumor site. In vitro mCAR showed dramatically reduced antigen binding and antigen-specific activation in the absence of proteases, but normal levels of binding and activity upon treatment with certain proteases. Masked CAR-T cells also showed antitumor efficacy in vivo comparable to that of unmasked CAR. Our study demonstrates the feasibility of improving the safety profile of conventional CARs and may also inspire future design of CAR molecules targeting broadly expressed TAAs. Copyright © 2017 The American Society of Gene and Cell Therapy. Published by Elsevier Inc. All rights reserved.

Antigenic mapping of an H9N2 avian influenza virus reveals two discrete antigenic sites and a novel mechanism of immune escape.

PubMed

Peacock, Thomas; Reddy, Kolli; James, Joe; Adamiak, Beata; Barclay, Wendy; Shelton, Holly; Iqbal, Munir

2016-01-07

H9N2 avian influenza virus is a major cause of poultry production loss across Asia leading to the wide use of vaccines. Efficacy of vaccines is often compromised due to the rapid emergence of antigenic variants. To improve the effectiveness of vaccines in the field, a better understanding of the antigenic epitopes of the major antigen, hemagglutinin, is required. To address this, a panel of nine monoclonal antibodies were generated against a contemporary Pakistani H9N2 isolate, which represents a major Asian H9N2 viral lineage. Antibodies were characterized in detail and used to select a total of 26 unique 'escape' mutants with substitutions across nine different amino acid residues in hemagglutinin including seven that have not been described as antigenic determinants for H9N2 viruses before. Competition assays and structural mapping revealed two novel, discrete antigenic sites "H9-A" and "H9-B". Additionally, a second subset of escape mutants contained amino acid deletions within the hemagglutinin receptor binding site. This constitutes a novel method of escape for group 1 hemagglutinins and could represent an alternative means for H9N2 viruses to overcome vaccine induced immunity. These results will guide surveillance efforts for arising antigenic variants as well as evidence based vaccine seed selection and vaccine design.

Antigenic mapping of an H9N2 avian influenza virus reveals two discrete antigenic sites and a novel mechanism of immune escape

PubMed Central

Peacock, Thomas; Reddy, Kolli; James, Joe; Adamiak, Beata; Barclay, Wendy; Shelton, Holly; Iqbal, Munir

2016-01-01

H9N2 avian influenza virus is a major cause of poultry production loss across Asia leading to the wide use of vaccines. Efficacy of vaccines is often compromised due to the rapid emergence of antigenic variants. To improve the effectiveness of vaccines in the field, a better understanding of the antigenic epitopes of the major antigen, hemagglutinin, is required. To address this, a panel of nine monoclonal antibodies were generated against a contemporary Pakistani H9N2 isolate, which represents a major Asian H9N2 viral lineage. Antibodies were characterized in detail and used to select a total of 26 unique ‘escape’ mutants with substitutions across nine different amino acid residues in hemagglutinin including seven that have not been described as antigenic determinants for H9N2 viruses before. Competition assays and structural mapping revealed two novel, discrete antigenic sites “H9-A” and “H9-B”. Additionally, a second subset of escape mutants contained amino acid deletions within the hemagglutinin receptor binding site. This constitutes a novel method of escape for group 1 hemagglutinins and could represent an alternative means for H9N2 viruses to overcome vaccine induced immunity. These results will guide surveillance efforts for arising antigenic variants as well as evidence based vaccine seed selection and vaccine design. PMID:26738561

Immunotherapy for Prostate Cancer: Lessons from Responses to Tumor-Associated Antigens

PubMed Central

Westdorp, Harm; Sköld, Annette E.; Snijer, Berit A.; Franik, Sebastian; Mulder, Sasja F.; Major, Pierre P.; Foley, Ronan; Gerritsen, Winald R.; de Vries, I. Jolanda M.

2014-01-01

Prostate cancer (PCa) is the most common cancer in men and the second most common cause of cancer-related death in men. In recent years, novel therapeutic options for PCa have been developed and studied extensively in clinical trials. Sipuleucel-T is the first cell-based immunotherapeutic vaccine for treatment of cancer. This vaccine consists of autologous mononuclear cells stimulated and loaded with an immunostimulatory fusion protein containing the prostate tumor antigen prostate acid posphatase. The choice of antigen might be key for the efficiency of cell-based immunotherapy. Depending on the treatment strategy, target antigens should be immunogenic, abundantly expressed by tumor cells, and preferably functionally important for the tumor to prevent loss of antigen expression. Autoimmune responses have been reported against several antigens expressed in the prostate, indicating that PCa is a suitable target for immunotherapy. In this review, we will discuss PCa antigens that exhibit immunogenic features and/or have been targeted in immunotherapeutic settings with promising results, and we highlight the hurdles and opportunities for cancer immunotherapy. PMID:24834066

Chemotherapy Enhances Cross-Presentation of Nuclear Tumor Antigens

PubMed Central

Anyaegbu, Chidozie C.; Lake, Richard A.; Heel, Kathy; Robinson, Bruce W.; Fisher, Scott A.

2014-01-01

Cross-presentation of tumor antigen is essential for efficient priming of naïve CD8+ T lymphocytes and induction of effective anti-tumor immunity. We hypothesized that the subcellular location of a tumor antigen could affect the efficiency of cross-presentation, and hence the outcome of anti-tumor responses to that antigen. We compared cross-presentation of a nominal antigen expressed in the nuclear, secretory, or cytoplasmic compartments of B16 melanoma tumors. All tumors expressed similar levels of the antigen. The antigen was cross-presented from all compartments but when the concentration was low, nuclear antigen was less efficiently cross-presented than antigen from other cellular locations. The efficiency of cross-presentation of the nuclear antigen was improved following chemotherapy-induced tumor cell apoptosis and this correlated with an increase in the proportion of effector CTL. These data demonstrate that chemotherapy improves nuclear tumor antigen cross-presentation and could be important for anti-cancer immunotherapies that target nuclear antigens. PMID:25243472

Lactoferrin for prevention of common viral infections.

PubMed

Wakabayashi, Hiroyuki; Oda, Hirotsugu; Yamauchi, Koji; Abe, Fumiaki

2014-11-01

Although lactoferrin has many biological functions, the host-protective effects against pathogenic microorganisms including bacteria, fungi, and viruses are regarded as one of the most important. Here, we review research on the protective role of lactoferrin administration against common viral infections. Many studies have shown the in vitro antiviral activity of lactoferrin against viral pathogens that cause common infections such as the common cold, influenza, gastroenteritis, summer cold, and herpes, where lactoferrin inhibits mainly viral attachment to the target cells. Recently, studies indicating the in vivo protective effects of lactoferrin by oral administration against common viral infections have been increasing. For instance, norovirus is an extremely important emerging human pathogen that causes a majority of gastroenteritis outbreaks worldwide that may be a target candidate for lactoferrin. Lactoferrin consumption reduced the incidence of noroviral gastroenteritis in children and a similar effect was observed in a wide range of ages in a preliminary survey. A recent in vitro study reported that lactoferrin inhibits both cellular attachment of the murine norovirus, a virus closely-related to the human norovirus, and viral replication in the cells by inducing antiviral cytokines interferon (IFN)-α/β. Lactoferrin administration also enhances NK cell activity and Th1 cytokine responses, which lead to protection against viral infections. In conclusion, lactoferrin consumption may protect the host from viral infections through inhibiting the attachment of a virus to the cells, replication of the virus in the cells, and enhancement of systemic immune functions. Copyright © 2014 Japanese Society of Chemotherapy and The Japanese Association for Infectious Diseases. Published by Elsevier Ltd. All rights reserved.

Novel anti-CD3 chimeric antigen receptor targeting of aggressive T cell malignancies

PubMed Central

Firor, Amelia E.; Pinz, Kevin G.; Jares, Alexander; Liu, Hua; Salman, Huda; Golightly, Marc; Lan, Fengshuo; Jiang, Xun; Ma, Yupo

2016-01-01

Peripheral T-cell lymphomas (PTCLS) comprise a diverse group of difficult to treat, very aggressive non-Hodgkin's lymphomas (NHLS) with poor prognoses and dismal patient outlook. Despite the fact that PTCLs comprise the majority of T-cell malignancies, the standard of care is poorly established. Chimeric antigen receptor (CAR) immunotherapy has shown in B-cell malignancies to be an effective curative option and this extends promise into treating T-cell malignancies. Because PTCLS frequently develop from mature T-cells, CD3 is similarly strongly and uniformly expressed in many PTCL malignancies, with expression specific to the hematological compartment thus making it an attractive target for CAR design. We engineered a robust 3rd generation anti-CD3 CAR construct (CD3CAR) into an NK cell line (NK-92). We found that CD3CAR NK-92 cells specifically and potently lysed diverse CD3+ human PTCL primary samples as well as T-cell leukemia cells lines ex vivo. Furthermore, CD3CAR NK-92 cells effectively controlled and suppressed Jurkat tumor cell growth in vivo and significantly prolonged survival. In this study, we present the CAR directed targeting of a novel target - CD3 using CAR modified NK-92 cells with an emphasis on efficacy, specificity, and potential for new therapeutic approaches that could improve the current standard of care for PTCLs. PMID:27494836

Burden and Seasonality of Viral Acute Respiratory Tract Infections among Outpatients in Southern Sri Lanka.

PubMed

Shapiro, David; Bodinayake, Champica K; Nagahawatte, Ajith; Devasiri, Vasantha; Kurukulasooriya, Ruvini; Hsiang, Jeremy; Nicholson, Bradley; De Silva, Aruna Dharshan; Østbye, Truls; Reller, Megan E; Woods, Christopher W; Tillekeratne, L Gayani

2017-07-01

In tropical and subtropical settings, the epidemiology of viral acute respiratory tract infections varies widely between countries. We determined the etiology, seasonality, and clinical presentation of viral acute respiratory tract infections among outpatients in southern Sri Lanka. From March 2013 to January 2015, we enrolled outpatients presenting with influenza-like illness (ILI). Nasal/nasopharyngeal samples were tested in duplicate using antigen-based rapid influenza testing and multiplex polymerase chain reaction (PCR) for respiratory viruses. Monthly proportion positive was calculated for each virus. Bivariable and multivariable logistic regression were used to identify associations between sociodemographic/clinical information and viral detection. Of 571 subjects, most (470, 82.3%) were ≥ 5 years of age and 53.1% were male. A respiratory virus was detected by PCR in 63.6% ( N = 363). Common viral etiologies included influenza (223, 39%), human enterovirus/rhinovirus (HEV/HRV, 14.5%), respiratory syncytial virus (RSV, 4.2%), and human metapneumovirus (hMPV, 3.9%). Both ILI and influenza showed clear seasonal variation, with peaks from March to June each year. RSV and hMPV activity peaked from May to July, whereas HEV/HRV was seen year-round. Patients with respiratory viruses detected were more likely to report pain with breathing (odds ratio [OR] = 2.60, P = 0.003), anorexia (OR = 2.29, P < 0.001), and fatigue (OR = 2.00, P = 0.002) compared with patients with no respiratory viruses detected. ILI showed clear seasonal variation in southern Sri Lanka, with most activity during March to June; peak activity was largely due to influenza. Targeted infection prevention activities such as influenza vaccination in January-February may have a large public health impact in this region.

Non-viral nucleic acid containing nanoparticles as cancer therapeutics

PubMed Central

Kozielski, Kristen L.; Rui, Yuan

2016-01-01

Introduction The delivery of nucleic acids such as DNA and short interfering RNA (siRNA) is promising for the treatment of many diseases, including cancer, by enabling novel biological mechanisms of action. Non-viral nanoparticles are a promising class of nucleic acid carriers that can be designed to be safer and more versatile than traditional viral vectors. Areas covered In this review, recent advances in the intracellular delivery of DNA and siRNA are described with a focus on non-viral nanoparticle-based delivery methods. Material properties that have enabled successful delivery are discussed as well as applications that have directly been applied to cancer therapy. Strategies to co-deliver different nucleic acids are highlighted, as are novel targets for nucleic acid co-delivery. Expert opinion The treatment of complex genetically-based diseases such as cancer can be enabled by safe and effective intracellular delivery of multiple nucleic acids. Non-viral nanoparticles can be fabricated to deliver multiple nucleic acids to the same cell simultaneously to prevent tumor cells from easily compensating for the knockdown or overexpression of one genetic target. The continued innovation of new therapeutic modalities and non-viral nanotechnologies to provide target-specific and personalized forms of gene therapy hold promise for genetic medicine to treat diseases like cancer in the clinic. PMID:27248202

Immune response to synthetic peptides representing antigenic sites on the glycoprotein of infectious hematopoietic necrosis virus

USGS Publications Warehouse

Emmenegger, Eveline J.; Huang, C.; LaPatra, S.; Winton, James R.

1995-01-01

Summary ― Monoclonal antibodies against infectious hematopoietic necrosis virus have been used to react with recombinant expression products in immunoblots and to select neutralization-resistant mutants for sequence analysis. These strategies identified neutralizing and non-neutralizing antigenic sites on the viral glycoprotein. Synthetic peptides based upon the amino acid sequences of these antigenic sites were synthesized and were injected together with an adjuvant into rainbow trout. The constructs generally failed to stimulate neutralizing antibodies in the fish. These results indicate that we need to understand more about the ability of peptide antigens to stimulate fish immune systems.

Preferential Targeting of Conserved Gag Regions after Vaccination with a Heterologous DNA Prime-Modified Vaccinia Virus Ankara Boost HIV-1 Vaccine Regimen.

PubMed

Bauer, Asli; Podola, Lilli; Mann, Philipp; Missanga, Marco; Haule, Antelmo; Sudi, Lwitiho; Nilsson, Charlotta; Kaluwa, Bahati; Lueer, Cornelia; Mwakatima, Maria; Munseri, Patricia J; Maboko, Leonard; Robb, Merlin L; Tovanabutra, Sodsai; Kijak, Gustavo; Marovich, Mary; McCormack, Sheena; Joseph, Sarah; Lyamuya, Eligius; Wahren, Britta; Sandström, Eric; Biberfeld, Gunnel; Hoelscher, Michael; Bakari, Muhammad; Kroidl, Arne; Geldmacher, Christof

2017-09-15

Prime-boost vaccination strategies against HIV-1 often include multiple variants for a given immunogen for better coverage of the extensive viral diversity. To study the immunologic effects of this approach, we characterized breadth, phenotype, function, and specificity of Gag-specific T cells induced by a DNA-prime modified vaccinia virus Ankara (MVA)-boost vaccination strategy, which uses mismatched Gag immunogens in the TamoVac 01 phase IIa trial. Healthy Tanzanian volunteers received three injections of the DNA-SMI vaccine encoding a subtype B and AB-recombinant Gag p37 and two vaccinations with MVA-CMDR encoding subtype A Gag p55 Gag-specific T-cell responses were studied in 42 vaccinees using fresh peripheral blood mononuclear cells. After the first MVA-CMDR boost, vaccine-induced gamma interferon-positive (IFN-γ + ) Gag-specific T-cell responses were dominated by CD4 + T cells ( P < 0.001 compared to CD8 + T cells) that coexpressed interleukin-2 (IL-2) (66.4%) and/or tumor necrosis factor alpha (TNF-α) (63.7%). A median of 3 antigenic regions were targeted with a higher-magnitude median response to Gag p24 regions, more conserved between prime and boost, compared to those of regions within Gag p15 (not primed) and Gag p17 (less conserved; P < 0.0001 for both). Four regions within Gag p24 each were targeted by 45% to 74% of vaccinees upon restimulation with DNA-SMI-Gag matched peptides. The response rate to individual antigenic regions correlated with the sequence homology between the MVA- and DNA Gag-encoded immunogens ( P = 0.04, r 2 = 0.47). In summary, after the first MVA-CMDR boost, the sequence-mismatched DNA-prime MVA-boost vaccine strategy induced a Gag-specific T-cell response that was dominated by polyfunctional CD4 + T cells and that targeted multiple antigenic regions within the conserved Gag p24 protein. IMPORTANCE Genetic diversity is a major challenge for the design of vaccines against variable viruses. While including multiple variants for a

Losartan and enalapril decrease viral absorption and interleukin 1 beta production by macrophages in an experimental dengue virus infection.

PubMed

Hernández-Fonseca, Juan Pablo; Durán, Anyelo; Valero, Nereida; Mosquera, Jesús

2015-11-01

The role of angiotensin II (Ang II) in dengue virus infection remains unknown. The aim of this study was to determine the effect of losartan, an antagonist of the angiotensin II type 1 receptor (AT1 receptor), and enalapril, an inhibitor of angiotensin I-converting enzyme (ACE), on viral antigen expression and IL-1β production in peritoneal macrophages infected with dengue virus type 2. Mice treated with losartan or enalapril and untreated controls were infected intraperitoneally with the virus, and macrophages were analyzed. Infection resulted in increased IL-1β production and a high percentage of cells expressing viral antigen, and this was decreased by treatment with anti-Ang II drugs, suggesting a role for Ang II in dengue virus infection.

Structural Relationships Between Minor and Major Proteins of Hepatitis B Surface Antigen

PubMed Central

Stibbe, Werner; Gerlich, Wolfram H.

1983-01-01

The minor glycoproteins from hepatitis B surface antigen, GP33 and GP36, contain at their carboxy-terminal part the sequence of the major protein P24. They have 55 additional amino acids at the amino-terminal part which are coded by the pre-S region of the viral DNA. Images PMID:6842680

Selective autophagy limits cauliflower mosaic virus infection by NBR1-mediated targeting of viral capsid protein and particles

PubMed Central

Hafrén, Anders; Macia, Jean-Luc; Love, Andrew J.; Milner, Joel J.; Drucker, Martin; Hofius, Daniel

2017-01-01

Autophagy plays a paramount role in mammalian antiviral immunity including direct targeting of viruses and their individual components, and many viruses have evolved measures to antagonize or even exploit autophagy mechanisms for the benefit of infection. In plants, however, the functions of autophagy in host immunity and viral pathogenesis are poorly understood. In this study, we have identified both anti- and proviral roles of autophagy in the compatible interaction of cauliflower mosaic virus (CaMV), a double-stranded DNA pararetrovirus, with the model plant Arabidopsis thaliana. We show that the autophagy cargo receptor NEIGHBOR OF BRCA1 (NBR1) targets nonassembled and virus particle-forming capsid proteins to mediate their autophagy-dependent degradation, thereby restricting the establishment of CaMV infection. Intriguingly, the CaMV-induced virus factory inclusions seem to protect against autophagic destruction by sequestering capsid proteins and coordinating particle assembly and storage. In addition, we found that virus-triggered autophagy prevents extensive senescence and tissue death of infected plants in a largely NBR1-independent manner. This survival function significantly extends the timespan of virus production, thereby increasing the chances for virus particle acquisition by aphid vectors and CaMV transmission. Together, our results provide evidence for the integration of selective autophagy into plant immunity against viruses and reveal potential viral strategies to evade and adapt autophagic processes for successful pathogenesis. PMID:28223514

Multiple chimeric antigen receptors successfully target chondroitin sulfate proteoglycan 4 in several different cancer histologies and cancer stem cells

PubMed Central

2014-01-01

Background The development of immunotherapy has led to significant progress in the treatment of metastatic cancer, including the development of genetic engineering technologies that redirect lymphocytes to recognize and target a wide variety of tumor antigens. Chimeric antigen receptors (CARs) are hybrid proteins combining antibody recognition domains linked to T cell signaling elements. Clinical trials of CAR-transduced peripheral blood lymphocytes (PBL) have induced remission of both solid organ and hematologic malignancies. Chondroitin sulfate proteoglycan 4 (CSPG4) is a promising target antigen that is overexpressed in multiple cancer histologies including melanoma, triple-negative breast cancer, glioblastoma, mesothelioma and sarcoma. Methods CSPG4 expression in cancer cell lines was assayed using flow cytometry (FACS) and reverse-transcription PCR (RT-PCR). Immunohistochemistry was utilized to assay resected melanomas and normal human tissues (n = 30) for CSPG4 expression and a reverse-phase protein array comprising 94 normal tissue samples was also interrogated for CSPG4 expression. CARs were successfully constructed from multiple murine antibodies (225.28S, TP41.2, 149.53) using second generation (CD28.CD3ζ) signaling domains. CAR sequences were cloned into a gamma-retroviral vector with subsequent successful production of retroviral supernatant and PBL transduction. CAR efficacy was assayed by cytokine release and cytolysis following coculture with target cell lines. Additionally, glioblastoma stem cells were generated from resected human tumors, and CSPG4 expression was determined by RT-PCR and FACS. Results Immunohistochemistry demonstrated prominent CSPG4 expression in melanoma tumors, but failed to demonstrate expression in any of the 30 normal human tissues studied. Two of 94 normal tissue protein lysates were positive by protein array. CAR constructs demonstrated cytokine secretion and cytolytic function after co-culture with tumor cell lines

Spacer length impacts the efficacy of targeted docetaxel conjugates in prostate-specific membrane antigen expressing prostate cancer.

PubMed

Peng, Zheng-Hong; Sima, Monika; Salama, Mohamed E; Kopečková, Pavla; Kopeček, Jindřich

2013-12-01

Combination of targeted delivery and controlled release is a powerful technique for cancer treatment. In this paper, we describe the design, synthesis, structure validation and biological properties of targeted and non-targeted N-(2-hydroxypropyl)methacrylamide (HPMA) copolymer-docetaxel conjugates. Docetaxel (DTX) was conjugated to HPMA copolymer via a tetrapeptide spacer (-GFLG-). 3-(1,3-dicarboxypropyl)-ureido]pentanedioic acid (DUPA) was used as the targeting moiety to actively deliver DTX for treatment of Prostate-Specific Membrane Antigen (PSMA) expressing prostate cancer. Short and long spacer DUPA monomers were prepared, and four HPMA copolymer--DTX conjugates (non-targeted, two targeted with short spacer of different molecular weight and targeted with long spacer) were prepared via Reversible Addition-Fragmentation Chain Transfer (RAFT) copolymerization. Following confirmation of PSMA expression on C4-2 cell line, the DTX conjugates' in vitro cytotoxicity was tested against C4-2 tumor cells and their anticancer efficacies were assessed in nude mice bearing s.c. human prostate adenocarcinoma C4-2 xenografts. The in vivo results show that the spacer length between targeting moieties and HPMA copolymer backbone can significantly affect the treatment efficacy of DTX conjugates against C4-2 tumor bearing nu/nu mice. Moreover, histological analysis indicated that the DUPA-targeted DTX conjugate with longer spacer had no toxicity in major organs of treated mice.

Spacer length impacts the efficacy of targeted docetaxel conjugates in prostate-specific membrane antigen expressing prostate cancer

PubMed Central

Peng, Zheng-Hong; Sima, Monika; Salama, Mohamed E.; Kopečková, Pavla; Kopeček, Jindřich

2015-01-01

Combination of targeted delivery and controlled release is a powerful technique for cancer treatment. In this paper, we describe the design, synthesis, structure validation and biological properties of targeted and non-targeted N-(2-hydroxypropyl)methacrylamide (HPMA) copolymer-docetaxel conjugates. Docetaxel (DTX) was conjugated to HPMA copolymer via a tetrapeptide spacer (–GFLG-). 3-(1,3-dicarboxypropyl)-ureido]pentanedioic acid (DUPA) was used as the targeting moiety to actively deliver DTX for treatment of Prostate-Specific Membrane Antigen (PSMA) expressing prostate cancer. Short and long spacer DUPA monomers were prepared, and four HPMA copolymer – DTX conjugates (non-targeted, two targeted with short spacer of different molecular weight and targeted with long spacer) were prepared via Reversible Addition-Fragmentation Chain Transfer (RAFT) copolymerization. Following confirmation of PSMA expression on C4-2 cell line, the DTX conjugates’ in vitro cytotoxicity was tested against C4-2 tumor cells and their anticancer efficacies were assessed in nude mice bearing s.c. human prostate adenocarcinoma C4-2 xenografts. The in vivo results show that the spacer length between targeting moieties and HPMA copolymer backbone can significantly affect the treatment efficacy of DTX conjugates against C4-2 tumor bearing nu/nu mice. Moreover, histological analysis indicated that the DUPA-targeted DTX conjugate with longer spacer had no toxicity in major organs of treated mice. PMID:24160903

Prostate-Specific Membrane Antigen Targeted Gold Nanoparticles for Theranostics of Prostate Cancer.

PubMed

Mangadlao, Joey Dacula; Wang, Xinning; McCleese, Christopher; Escamilla, Maria; Ramamurthy, Gopalakrishnan; Wang, Ziying; Govande, Mukul; Basilion, James P; Burda, Clemens

2018-04-24

Prostate cancer is one of the most common cancers and among the leading causes of cancer deaths in the United States. Men diagnosed with the disease typically undergo radical prostatectomy, which often results in incontinence and impotence. Recurrence of the disease is often experienced by most patients with incomplete prostatectomy during surgery. Hence, the development of a technique that will enable surgeons to achieve a more precise prostatectomy remains an open challenge. In this contribution, we report a theranostic agent (AuNP-5kPEG-PSMA-1-Pc4) based on prostate-specific membrane antigen (PSMA-1)-targeted gold nanoparticles (AuNPs) loaded with a fluorescent photodynamic therapy (PDT) drug, Pc4. The fabricated nanoparticles are well-characterized by spectroscopic and imaging techniques and are found to be stable over a wide range of solvents, buffers, and media. In vitro cellular uptake experiments demonstrated significantly higher nanoparticle uptake in PSMA-positive PC3pip cells than in PSMA-negative PC3flu cells. Further, more complete cell killing was observed in Pc3pip than in PC3flu cells upon exposure to light at different doses, demonstrating active targeting followed by Pc4 delivery. Likewise, in vivo studies showed remission on PSMA-expressing tumors 14 days post-PDT. Atomic absorption spectroscopy revealed that targeted AuNPs accumulate 4-fold higher in PC3pip than in PC3flu tumors. The nanoparticle system described herein is envisioned to provide surgical guidance for prostate tumor resection and therapeutic intervention when surgery is insufficient.

Multiplex diagnosis of viral infectious diseases (AIDS, hepatitis C, and hepatitis A) based on point of care lateral flow assay using engineered proteinticles.

PubMed

Lee, Jong-Hwan; Seo, Hyuk Seong; Kwon, Jung-Hyuk; Kim, Hee-Tae; Kwon, Koo Chul; Sim, Sang Jun; Cha, Young Joo; Lee, Jeewon

2015-07-15

Lateral flow assay (LFA) is an attractive method for rapid, simple, and cost-effective point of care diagnosis. For LFA-based multiplex diagnosis of three viral intractable diseases (acquired immune deficiency syndrome and hepatitis C and A), here we developed proteinticle-based 7 different 3D probes that display different viral antigens on their surface, which were synthesized in Escherichia coli by self-assembly of human ferritin heavy chain that was already engineered by genetically linking viral antigens to its C-terminus. Each of the three test lines on LFA strip contains the proteinticle probes to detect disease-specific anti-viral antibodies. Compared to peptide probes, the proteinticle probes were evidently more sensitive, and the proteinticle probe-based LFA successfully diagnosed all the 20 patient sera per each disease without a false negative signal, whereas the diagnostic sensitivities in the peptide probe-based LFAs were 65-90%. Duplex and triplex assays performed with randomly mixed patient sera gave only true positive signals for all the 20 serum mixtures without any false positive signals, indicating 100% sensitivity and 100% specificity. It seems that on the proteinticle surface the antigenic peptides have homogeneous orientation and conformation without inter-peptide clustering and hence lead to the enhanced diagnostic performance with solving the problems of traditional diagnostic probes. Although the multiplex diagnosis of three viral diseases above was demonstrated as proof-of-concept here, the proposed LFA system can be applied to multiplex point of care diagnosis of other intractable diseases. Copyright © 2015 Elsevier B.V. All rights reserved.

Immunotherapy of Malignant Disease Using Chimeric Antigen Receptor Engrafted T Cells

PubMed Central

Maher, John

2012-01-01

Chimeric antigen receptor- (CAR-) based immunotherapy has been under development for almost 25 years, over which period it has progressed from a new but cumbersome technology to an emerging therapeutic modality for malignant disease. The approach involves the genetic engineering of fusion receptors (CARs) that couple the HLA-independent binding of cell surface target molecules to the delivery of a tailored activating signal to host immune cells. Engineered CARs are delivered most commonly to peripheral blood T cells using a range of vector systems, most commonly integrating viral vectors. Preclinical refinement of this approach has proceeded over several years to the point that clinical testing is now being undertaken at several centres, using increasingly sophisticated and therapeutically successful genetic payloads. This paper considers several aspects of the pre-clinical and clinical development of CAR-based immunotherapy and how this technology is acquiring an increasing niche in the treatment of both solid and haematological malignancies. PMID:23304553

Multiplex Degenerate Primer Design for Targeted Whole Genome Amplification of Many Viral Genomes

DOE Office of Scientific and Technical Information (OSTI.GOV)

Gardner, Shea N.; Jaing, Crystal J.; Elsheikh, Maher M.

Background . Targeted enrichment improves coverage of highly mutable viruses at low concentration in complex samples. Degenerate primers that anneal to conserved regions can facilitate amplification of divergent, low concentration variants, even when the strain present is unknown. Results . A tool for designing multiplex sets of degenerate sequencing primers to tile overlapping amplicons across multiple whole genomes is described. The new script, run_tiled_primers, is part of the PriMux software. Primers were designed for each segment of South American hemorrhagic fever viruses, tick-borne encephalitis, Henipaviruses, Arenaviruses, Filoviruses, Crimean-Congo hemorrhagic fever virus, Rift Valley fever virus, and Japanese encephalitis virus. Eachmore » group is highly diverse with as little as 5% genome consensus. Primer sets were computationally checked for nontarget cross reactions against the NCBI nucleotide sequence database. Primers for murine hepatitis virus were demonstrated in the lab to specifically amplify selected genes from a laboratory cultured strain that had undergone extensive passage in vitro and in vivo. Conclusions . This software should help researchers design multiplex sets of primers for targeted whole genome enrichment prior to sequencing to obtain better coverage of low titer, divergent viruses. Applications include viral discovery from a complex background and improved sensitivity and coverage of rapidly evolving strains or variants in a gene family.« less

Multiplex Degenerate Primer Design for Targeted Whole Genome Amplification of Many Viral Genomes

DOE PAGES

Gardner, Shea N.; Jaing, Crystal J.; Elsheikh, Maher M.; ...

2014-01-01

Background . Targeted enrichment improves coverage of highly mutable viruses at low concentration in complex samples. Degenerate primers that anneal to conserved regions can facilitate amplification of divergent, low concentration variants, even when the strain present is unknown. Results . A tool for designing multiplex sets of degenerate sequencing primers to tile overlapping amplicons across multiple whole genomes is described. The new script, run_tiled_primers, is part of the PriMux software. Primers were designed for each segment of South American hemorrhagic fever viruses, tick-borne encephalitis, Henipaviruses, Arenaviruses, Filoviruses, Crimean-Congo hemorrhagic fever virus, Rift Valley fever virus, and Japanese encephalitis virus. Eachmore » group is highly diverse with as little as 5% genome consensus. Primer sets were computationally checked for nontarget cross reactions against the NCBI nucleotide sequence database. Primers for murine hepatitis virus were demonstrated in the lab to specifically amplify selected genes from a laboratory cultured strain that had undergone extensive passage in vitro and in vivo. Conclusions . This software should help researchers design multiplex sets of primers for targeted whole genome enrichment prior to sequencing to obtain better coverage of low titer, divergent viruses. Applications include viral discovery from a complex background and improved sensitivity and coverage of rapidly evolving strains or variants in a gene family.« less

A host-restricted viral vector for antigen-specific immunization against Lyme disease pathogen.

PubMed

Xiao, Sa; Kumar, Manish; Yang, Xiuli; Akkoyunlu, Mustafa; Collins, Peter L; Samal, Siba K; Pal, Utpal

2011-07-18

Newcastle disease virus (NDV) is an avian virus that is attenuated in primates and is a potential vaccine vector for human use. We evaluated NDV as a vector for expressing selected antigens of the Lyme disease pathogen Borrelia burgdorferi. A series of recombinant NDVs were generated that expressed intracellular or extracellular forms of two B. burgdorferi antigens: namely, the basic membrane protein A (BmpA) and the outer surface protein C (OspC). Expression of the intracellular and extracellular forms of these antigens was confirmed in cultured chicken cells. C3H or Balb/C mice that were immunized intranasally with the NDV vectors mounted vigorous serum antibody responses against the NDV vector, but failed to mount a robust response against either the intracellular or extracellular forms of BmpA or OspC. By contrast, a single immunization of hamsters with the NDV vectors via the intranasal, intramuscular, or intraperitoneal route resulted in rapid and rigorous antibody responses against the intracellular or extracellular forms of BmpA and OspC. When groups of hamsters were separately inoculated with various NDV vectors and challenged with B. burgdorferi (10(8)cells/animal), immunization with vector expressing either intracellular or extracellular BmpA was associated with a significant reduction of the pathogen load in the joints. Taken together, our studies highlighted the importance of NDV as vaccine vector that can be used for simple yet effective immunization of hosts against bacterial infections including Lyme disease. Copyright © 2011 Elsevier Ltd. All rights reserved.

[Viral hepatitis A and B in anthropoid apes of the Moscow Zoo].

PubMed

Anan'ev, V A; Viazov, S O; Garanina, N M; Doroshenko, N V; Zhilina, N N

1984-01-01

The rate of occurrence of hepatitis A and B virus specific markers in anthropoid apes of the Moscow Zoo (3 chimpanzees, 3 gorillas, 8 orang-outangs) was studied. Long-term persistence of HBs-antigen in orang-outang accompanied by the presence of HBe-antigen and anti-HBs was demonstrated. The presence of anti-HBs in gorillas and orang-outangs was recorded. Antibodies to hepatitis A virus were found in all the animal species examined. Cases of acute virus hepatitis A in orang-outangs are described contracted from the personnel handling the animals and suffering from this type of hepatitis. Morphological features of the course of hepatitis A in the presence of HBs-antigen carrier state are described. The possibility of active immunization of susceptible animals and the personnel of the zoo against viral hepatitides is discussed.

Proliferating cell nuclear antigen (Pcna) as a direct downstream target gene of Hoxc8

DOE Office of Scientific and Technical Information (OSTI.GOV)

Min, Hyehyun; Lee, Ji-Yeon; Bok, Jinwoong

2010-02-19

Hoxc8 is a member of Hox family transcription factors that play crucial roles in spatiotemporal body patterning during embryogenesis. Hox proteins contain a conserved 61 amino acid homeodomain, which is responsible for recognition and binding of the proteins onto Hox-specific DNA binding motifs and regulates expression of their target genes. Previously, using proteome analysis, we identified Proliferating cell nuclear antigen (Pcna) as one of the putative target genes of Hoxc8. Here, we asked whether Hoxc8 regulates Pcna expression by directly binding to the regulatory sequence of Pcna. In mouse embryos at embryonic day 11.5, the expression pattern of Pcna wasmore » similar to that of Hoxc8 along the anteroposterior body axis. Moreover, Pcna transcript levels as well as cell proliferation rate were increased by overexpression of Hoxc8 in C3H10T1/2 mouse embryonic fibroblast cells. Characterization of 2.3 kb genomic sequence upstream of Pcna coding region revealed that the upstream sequence contains several Hox core binding sequences and one Hox-Pbx binding sequence. Direct binding of Hoxc8 proteins to the Pcna regulatory sequence was verified by chromatin immunoprecipitation assay. Taken together, our data suggest that Pcna is a direct downstream target of Hoxc8.« less

Viral and Cellular Determinants of the Hepatitis C Virus Envelope-Heparan Sulfate Interaction▿

PubMed Central

Barth, Heidi; Schnober, Eva K.; Zhang, Fuming; Linhardt, Robert J.; Depla, Erik; Boson, Bertrand; Cosset, Francois-Loic; Patel, Arvind H.; Blum, Hubert E.; Baumert, Thomas F.

2006-01-01

Cellular binding and entry of hepatitis C virus (HCV) are the first steps of viral infection and represent a major target for antiviral antibodies and novel therapeutic strategies. We have recently demonstrated that heparan sulfate (HS) plays a key role in the binding of HCV envelope glycoprotein E2 to target cells (Barth et al., J. Biol. Chem. 278:41003-41012, 2003). In this study, we characterized the HCV-HS interaction and analyzed its inhibition by antiviral host immune responses. Using recombinant envelope glycoproteins, virus-like particles, and HCV pseudoparticles as model systems for the early steps of viral infection, we mapped viral and cellular determinants of HCV-HS interaction. HCV-HS binding required a specific HS structure that included N-sulfo groups and a minimum of 10 to 14 saccharide subunits. HCV envelope binding to HS was mediated by four viral epitopes overlapping the E2 hypervariable region 1 and E2-CD81 binding domains. In functional studies using HCV pseudoparticles, we demonstrate that HCV binding and entry are specifically inhibited by highly sulfated HS. Finally, HCV-HS binding was markedly inhibited by antiviral antibodies derived from HCV-infected individuals. In conclusion, our results demonstrate that binding of the viral envelope to a specific HS configuration represents an important step for the initiation of viral infection and is a target of antiviral host immune responses in vivo. Mapping of viral and cellular determinants of HCV-HS interaction sets the stage for the development of novel HS-based antiviral strategies targeting viral attachment and entry. PMID:16928753

Information Overload and Viral Marketing: Countermeasures and Strategies

NASA Astrophysics Data System (ADS)

Cheng, Jiesi; Sun, Aaron; Zeng, Daniel

Studying information diffusion through social networks has become an active research topic with important implications in viral marketing applications. One of the fundamental algorithmic problems related to viral marketing is the Influence Maximization (IM) problem: given an social network, which set of nodes should be considered by the viral marketer as the initial targets, in order to maximize the influence of the advertising message. In this work, we study the IM problem in an information-overloaded online social network. Information overload occurs when individuals receive more information than they can process, which can cause negative impacts on the overall marketing effectiveness. Many practical countermeasures have been proposed for alleviating the load of information on recipients. However, how these approaches can benefit viral marketers is not well understood. In our work, we have adapted the classic Information Cascade Model to incorporate information overload and study its countermeasures. Our results suggest that effective control of information overload has the potential to improve marketing effectiveness, but the targeting strategy should be re-designed in response to these countermeasures.

Epstein-Barr virus nuclear antigen EBNA-LP is essential for transforming naïve B cells, and facilitates recruitment of transcription factors to the viral genome.

PubMed

Szymula, Agnieszka; Palermo, Richard D; Bayoumy, Amr; Groves, Ian J; Ba Abdullah, Mohammed; Holder, Beth; White, Robert E

2018-02-01

The Epstein-Barr virus (EBV) nuclear antigen leader protein (EBNA-LP) is the first viral latency-associated protein produced after EBV infection of resting B cells. Its role in B cell transformation is poorly defined, but it has been reported to enhance gene activation by the EBV protein EBNA2 in vitro. We generated EBNA-LP knockout (LPKO) EBVs containing a STOP codon within each repeat unit of internal repeat 1 (IR1). EBNA-LP-mutant EBVs established lymphoblastoid cell lines (LCLs) from adult B cells at reduced efficiency, but not from umbilical cord B cells, which died approximately two weeks after infection. Adult B cells only established EBNA-LP-null LCLs with a memory (CD27+) phenotype. Quantitative PCR analysis of virus gene expression after infection identified both an altered ratio of the EBNA genes, and a dramatic reduction in transcript levels of both EBNA2-regulated virus genes (LMP1 and LMP2) and the EBNA2-independent EBER genes in the first 2 weeks. By 30 days post infection, LPKO transcription was the same as wild-type EBV. In contrast, EBNA2-regulated cellular genes were induced efficiently by LPKO viruses. Chromatin immunoprecipitation revealed that EBNA2 and the host transcription factors EBF1 and RBPJ were delayed in their recruitment to all viral latency promoters tested, whereas these same factors were recruited efficiently to several host genes, which exhibited increased EBNA2 recruitment. We conclude that EBNA-LP does not simply co-operate with EBNA2 in activating gene transcription, but rather facilitates the recruitment of several transcription factors to the viral genome, to enable transcription of virus latency genes. Additionally, our findings suggest that EBNA-LP is essential for the survival of EBV-infected naïve B cells.

Epstein-Barr virus nuclear antigen EBNA-LP is essential for transforming naïve B cells, and facilitates recruitment of transcription factors to the viral genome

PubMed Central

Szymula, Agnieszka; Palermo, Richard D.; Bayoumy, Amr; Groves, Ian J.

2018-01-01

The Epstein-Barr virus (EBV) nuclear antigen leader protein (EBNA-LP) is the first viral latency-associated protein produced after EBV infection of resting B cells. Its role in B cell transformation is poorly defined, but it has been reported to enhance gene activation by the EBV protein EBNA2 in vitro. We generated EBNA-LP knockout (LPKO) EBVs containing a STOP codon within each repeat unit of internal repeat 1 (IR1). EBNA-LP-mutant EBVs established lymphoblastoid cell lines (LCLs) from adult B cells at reduced efficiency, but not from umbilical cord B cells, which died approximately two weeks after infection. Adult B cells only established EBNA-LP-null LCLs with a memory (CD27+) phenotype. Quantitative PCR analysis of virus gene expression after infection identified both an altered ratio of the EBNA genes, and a dramatic reduction in transcript levels of both EBNA2-regulated virus genes (LMP1 and LMP2) and the EBNA2-independent EBER genes in the first 2 weeks. By 30 days post infection, LPKO transcription was the same as wild-type EBV. In contrast, EBNA2-regulated cellular genes were induced efficiently by LPKO viruses. Chromatin immunoprecipitation revealed that EBNA2 and the host transcription factors EBF1 and RBPJ were delayed in their recruitment to all viral latency promoters tested, whereas these same factors were recruited efficiently to several host genes, which exhibited increased EBNA2 recruitment. We conclude that EBNA-LP does not simply co-operate with EBNA2 in activating gene transcription, but rather facilitates the recruitment of several transcription factors to the viral genome, to enable transcription of virus latency genes. Additionally, our findings suggest that EBNA-LP is essential for the survival of EBV-infected naïve B cells. PMID:29462212

Merkel Cell Polyomavirus Large T Antigen Has Growth-Promoting and Inhibitory Activities

PubMed Central

Cheng, Jingwei; Rozenblatt-Rosen, Orit; Paulson, Kelly G.; Nghiem, Paul

2013-01-01

Merkel cell carcinoma (MCC) is a rare and aggressive form of skin cancer. In at least 80% of all MCC, Merkel cell polyomavirus (MCPyV) DNA has undergone clonal integration into the host cell genome, and most tumors express the MCPyV large and small T antigens. In all cases of MCC reported to date, the integrated MCPyV genome has undergone mutations in the large T antigen. These mutations result in expression of a truncated large T antigen that retains the Rb binding or LXCXE motif but deletes the DNA binding and helicase domains. However, the transforming functions of full-length and truncated MCPyV large T antigen are unknown. We compared the transforming activities of full-length, truncated, and alternatively spliced 57kT forms of MCPyV large T antigen. MCPyV large T antigen could bind to Rb but was unable to bind to p53. Furthermore, MCPyV-truncated large T antigen was more effective than full-length and 57kT large T antigen in promoting the growth of human and mouse fibroblasts. In contrast, expression of the MCPyV large T antigen C-terminal 100 residues could inhibit the growth of several different cell types. These data imply that the deletion of the C terminus of MCPyV large T antigen found in MCC serves not only to disrupt viral replication but also results in the loss of a distinct growth-inhibitory function intrinsic to this region. PMID:23514892

Broad-spectrum antivirals against viral fusion

PubMed Central

Vigant, Frederic; Santos, Nuno C.; Lee, Benhur

2015-01-01

Effective antivirals have been developed against specific viruses, such as HIV, Hepatitis C virus and influenza virus. This ‘one bug–one drug’ approach to antiviral drug development can be successful, but it may be inadequate for responding to an increasing diversity of viruses that cause significant diseases in humans. The majority of viral pathogens that cause emerging and re-emerging infectious diseases are membrane-enveloped viruses, which require the fusion of viral and cell membranes for virus entry. Therefore, antivirals that target the membrane fusion process represent new paradigms for broad-spectrum antiviral discovery. In this Review, we discuss the mechanisms responsible for the fusion between virus and cell membranes and explore how broad-spectrum antivirals target this process to prevent virus entry. PMID:26075364

In vitro models of viral-induced congenital deafness.

PubMed

Davis, G L

1981-10-01

Cytomegalovirus (CMV) infects 1 to 2 percent of liveborn infants in the United States and causes varying degrees of perceptive hearing loss. There are eight reported pathologic studies of temporal bones in CMV-infected neonates. Viral replication occurs in nonsensory endolabyrinthine epithelium, but viral antigen is also found in the organ of Corti and spiral ganglion neurons, and CMV has been cultured from perilymph. Further clinicopathologic correlation is frustrated, since the inner ear cannot be biopsied during life, and the number of temporal bones available for study is limited, owing to the decrease in the number of autopsies being performed. Inoculation of CMV into newborn mice, and extracorporeal preparations of mouse and guinea pig fetal inner ears, either in organ culture or as grafts on chick chorioallantoic membranes, yields viral perilabyrinthitis. The different ultrastructural appearances of CMV replicating in epithelial and mesenchymal cells show that animal CMV replicates in mesenchymal cells and human CMV replicates in epithelial cells of the inner ear. These different ultrastructural patterns indicate that the chromophobe (transitional) cells of the stria vascularis of the guinea pig are of mesenchymal origin.

Laboratory evolution of artificially expanded DNA gives redesignable aptamers that target the toxic form of anthrax protective antigen

PubMed Central

Biondi, Elisa; Lane, Joshua D.; Das, Debasis; Dasgupta, Saurja; Piccirilli, Joseph A.; Hoshika, Shuichi; Bradley, Kevin M.; Krantz, Bryan A.; Benner, Steven A.

2016-01-01

Reported here is a laboratory in vitro evolution (LIVE) experiment based on an artificially expanded genetic information system (AEGIS). This experiment delivers the first example of an AEGIS aptamer that binds to an isolated protein target, the first whose structural contact with its target has been outlined and the first to inhibit biologically important activities of its target, the protective antigen from Bacillus anthracis. We show how rational design based on secondary structure predictions can also direct the use of AEGIS to improve the stability and binding of the aptamer to its target. The final aptamer has a dissociation constant of ∼35 nM. These results illustrate the value of AEGIS-LIVE for those seeking to obtain receptors and ligands without the complexities of medicinal chemistry, and also challenge the biophysical community to develop new tools to analyze the spectroscopic signatures of new DNA folds that will emerge in synthetic genetic systems replacing standard DNA and RNA as platforms for LIVE. PMID:27701076

Transgenic antigen-specific, HLA-A*02:01-allo-restricted cytotoxic T cells recognize tumor-associated target antigen STEAP1 with high specificity

PubMed Central

Schirmer, David; Grünewald, Thomas G. P.; Klar, Richard; Schmidt, Oxana; Wohlleber, Dirk; Rubío, Rebeca Alba; Uckert, Wolfgang; Thiel, Uwe; Bohne, Felix; Busch, Dirk H.; Krackhardt, Angela M.; Burdach, Stefan; Richter, Günther H. S.

2016-01-01

ABSTRACT Pediatric cancers, including Ewing sarcoma (ES), are only weakly immunogenic and the tumor-patients' immune system often is devoid of effector T cells for tumor elimination. Based on expression profiling technology, targetable tumor-associated antigens (TAA) are identified and exploited for engineered T-cell therapy. Here, the specific recognition and lytic potential of transgenic allo-restricted CD8+ T cells, directed against the ES-associated antigen 6-transmembrane epithelial antigen of the prostate 1 (STEAP1), was examined. Following repetitive STEAP1130 peptide-driven stimulations with HLA-A*02:01+ dendritic cells (DC), allo-restricted HLA-A*02:01− CD8+ T cells were sorted with HLA-A*02:01/peptide multimers and expanded by limiting dilution. After functional analysis of suitable T cell clones via ELISpot, flow cytometry and xCELLigence assay, T cell receptors' (TCR) α- and β-chains were identified, cloned into retroviral vectors, codon optimized, transfected into HLA-A*02:01− primary T cell populations and tested again for specificity and lytic capacity in vitro and in a Rag2−/−γc−/− mouse model. Initially generated transgenic T cells specifically recognized STEAP1130-pulsed or transfected cells in the context of HLA-A*02:01 with minimal cross-reactivity as determined by specific interferon-γ (IFNγ) release, lysed cells and inhibited growth of HLA-A*02:01+ ES lines more effectively than HLA-A*02:01− ES lines. In vivo tumor growth was inhibited more effectively with transgenic STEAP1130-specific T cells than with unspecific T cells. Our results identify TCRs capable of recognizing and inhibiting growth of STEAP1-expressing HLA-A*02:01+ ES cells in vitro and in vivo in a highly restricted manner. As STEAP1 is overexpressed in a wide variety of cancers, we anticipate these STEAP1-specific TCRs to be potentially useful for immunotherapy of other STEAP1-expressing tumors. PMID:27471654

Chimeric antigen receptor (CAR)-directed adoptive immunotherapy: a new era in targeted cancer therapy.

PubMed

Chen, Yamei; Liu, Delong

2014-01-01

As a result of the recent advances in molecular immunology, virology, genetics, and cell processing, chimeric antigen receptor (CAR)-directed cancer therapy has finally arrived for clinical application. CAR-directed adoptive immunotherapy represents a novel form of gene therapy, cellular therapy, and immunotherapy, a combination of three in one. Early phase clinical trial was reported in patients with refractory chronic lymphoid leukemia with 17p deletion. Accompanying the cytokine storm and tumor lysis syndrome was the shocking disappearance of the leukemia cells refractory to chemotherapy and monoclonal antibodies. CAR therapy was reproduced in both children and adults with refractory acute lymphoid leukemia. The CAR technology is being explored for solid tumor therapy, such as glioma. Close to 30 clinical trials are underway in the related fields (www.clinicaltrials.gov). Further improvement in gene targeting, cell expansion, delivery constructs (such as using Sleeping Beauty or Piggyback transposons) will undoubtedly enhance clinical utility. It is foreseeable that CAR-engineered T cell therapy will bring targeted cancer therapy into a new era.

A novel multi-antigen virally vectored vaccine against Mycobacterium avium subspecies paratuberculosis.

PubMed

Bull, Tim J; Gilbert, Sarah C; Sridhar, Saranya; Linedale, Richard; Dierkes, Nicola; Sidi-Boumedine, Karim; Hermon-Taylor, John

2007-11-28

Mycobacterium avium subspecies paratuberculosis causes systemic infection and chronic intestinal inflammation in many species including primates. Humans are exposed through milk and from sources of environmental contamination. Hitherto, the only vaccines available against Mycobacterium avium subspecies paratuberculosis have been limited to veterinary use and comprised attenuated or killed organisms. We developed a vaccine comprising a fusion construct designated HAV, containing components of two secreted and two cell surface Mycobacterium avium subspecies paratuberculosis proteins. HAV was transformed into DNA, human Adenovirus 5 (Ad5) and Modified Vaccinia Ankara (MVA) delivery vectors. Full length expression of the predicted 95 kDa fusion protein was confirmed. Vaccination of naïve and Mycobacterium avium subspecies paratuberculosis infected C57BL/6 mice using DNA-prime/MVA-boost or Ad5-prime/MVA-boost protocols was highly immunogenic resulting in significant IFN-gamma ELISPOT responses by splenocytes against recombinant vaccine antigens and a range of HAV specific peptides. This included strong recognition of a T-cell epitope GFAEINPIA located near the C-terminus of the fusion protein. Antibody responses to recombinant vaccine antigens and HAV specific peptides but not GFAEINPIA, also occurred. No immune recognition of vaccine antigens occurred in any sham vaccinated Mycobacterium avium subspecies paratuberculosis infected mice. Vaccination using either protocol significantly attenuated pre-existing Mycobacterium avium subspecies paratuberculosis infection measured by qPCR in spleen and liver and the Ad5-prime/MVA-boost protocol also conferred some protection against subsequent challenge. No adverse effects of vaccination occurred in any of the mice. A range of modern veterinary and clinical vaccines for the treatment and prevention of disease caused by Mycobacterium avium subspecies paratuberculosis are needed. The present vaccine proved to be highly

Nanoparticle-based B-cell targeting vaccines: Tailoring of humoral immune responses by functionalization with different TLR-ligands.

PubMed

Zilker, Claudia; Kozlova, Diana; Sokolova, Viktoriya; Yan, Huimin; Epple, Matthias; Überla, Klaus; Temchura, Vladimir

2017-01-01

Induction of an appropriate type of humoral immune response during vaccination is essential for protection against viral and bacterial infections. We recently observed that biodegradable calcium phosphate (CaP) nanoparticles coated with proteins efficiently targeted and activated naïve antigen-specific B-cells in vitro. We now compared different administration routes for CaP-nanoparticles and demonstrated that intramuscular immunization with such CaP-nanoparticles induced stronger immune responses than immunization with monovalent antigen. Additional functionalization of the CaP-nanoparticles with TRL-ligands allowed modulating the IgG subtype response and the level of mucosal IgA antibodies. CpG-containing CaP-nanoparticles were as immunogenic as a virus-like particle vaccine. Functionalization of CaP-nanoparticles with T-helper cell epitopes or CpG also allowed overcoming lack of T-cell help. Thus, our results indicate that CaP-nanoparticle-based B-cell targeting vaccines functionalized with TLR-ligands can serve as a versatile platform for efficient induction and modulation of humoral immune responses in vivo. Copyright © 2016 Elsevier Inc. All rights reserved.

Localization of latency-associated nuclear antigen (LANA) on mitotic chromosomes

DOE Office of Scientific and Technical Information (OSTI.GOV)

Rahayu, Retno; Ohsaki, Eriko; Omori, Hiroko