Intrabodies targeting the Kaposi sarcoma–associated herpesvirus latency antigen inhibit viral persistence in lymphoma cells

Kaposi sarcoma–associated herpesvirus (KSHV) latency-associated nuclear antigen-1 (LANA1) is essential for the maintenance and segregation of viral episomes in KSHV latently infected B cells. We report development of intracellular, rabbit-derived antibodies generated by phage display technology, which bind to N-terminal LANA1 epitopes and neutralize the chromosome-binding activity of LANA1. Although these cloned single-chain variable fragments (scFvs) show relatively low binding affinities for the LANA1 viral antigen in in vitro assays, they nonetheless outcompete KSHV-seropositive human sera for LANA1 epitope binding. In heterologous cells, intracellular intrabody expression inhibits LANA1-dependent plasmid maintenance of both an artificial plasmid containing KSHV LANA1 binding sequences and a bacterial artificial chromosome containing the entire KSHV genome. In KSHV naturally infected primary effusion lymphoma cells, intracellular intrabody expression causes a reduction or loss of the typical LANA1 punctate, nuclear pattern. This morphologically apparent LANA1 dispersion correlates to loss of viral episome by molecular analysis. These data suggest a novel approach to antiherpes viral therapy and confirm LANA1 is critical target for neutralization of KSHV viral latency.

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In Vitro Characterization of Neutralizing Hen Antibodies to Coxsackievirus A16

International Journal of Molecular Sciences ◽

10.3390/ijms22084146 ◽

2021 ◽

Vol 22 (8) ◽

pp. 4146

Author(s):

Pharaoh Fellow Mwale ◽

Chi-Hsin Lee ◽

Peng-Nien Huang ◽

Sung-Nien Tseng ◽

Shin-Ru Shih ◽

...

Keyword(s):

Viral Infection ◽

Clinical Symptoms ◽

Binding Protein ◽

Foot And Mouth Disease ◽

Binding Activity ◽

In Vitro Assays ◽

Single Chain ◽

Coxsackievirus A16 ◽

Causative Agents

Coxsackievirus A16 (CA16) is one of the major causative agents of hand, foot, and mouth disease (HFMD). Children aged <5 years are the most affected by CA16 HFMD globally. Although clinical symptoms of CA16 infections are usually mild, severe complications, such as aseptic meningitis or even death, have been recorded. Currently, no vaccine or antiviral therapy for CA16 infection exists. Single-chain variable fragment (scFv) antibodies significantly inhibit viral infection and could be a potential treatment for controlling the infection. In this study, scFv phage display libraries were constructed from splenocytes of a laying hen immunized with CA16-infected lysate. The pComb3X vector containing the scFv genes was introduced into ER2738 Escherichia coli and rescued by helper phages to express scFv molecules. After screening with five cycles of bio-panning, an effective scFv antibody showing favorable binding activity to proteins in CA16-infected lysate on ELISA plates was selected. Importantly, the selected scFv clone showed a neutralizing capability against the CA16 virus and cross-reacted with viral proteins in EV71-infected lysate. Intriguingly, polyclonal IgY antibody not only showed binding specificity against proteins in CA16-infected lysate but also showed significant neutralization activities. Nevertheless, IgY-binding protein did not cross-react with proteins in EV71-infected lysate. These results suggest that the IgY- and scFv-binding protein antibodies provide protection against CA16 viral infection in in vitro assays and may be potential candidates for treating CA16 infection in vulnerable young children.

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Hippuristanol Reduces the Viability of Primary Effusion Lymphoma Cells both in Vitro and in Vivo

Marine Drugs ◽

10.3390/md11093410 ◽

2013 ◽

Vol 11 (9) ◽

pp. 3410-3424 ◽

Cited By ~ 11

Author(s):

Chie Ishikawa ◽

Junichi Tanaka ◽

Harutaka Katano ◽

Masachika Senba ◽

Naoki Mori

Keyword(s):

Primary Effusion Lymphoma ◽

Lymphoma Cells

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Engineered Fragments of the PSMA-Specific 5D3 Antibody and Their Functional Characterization

International Journal of Molecular Sciences ◽

10.3390/ijms21186672 ◽

2020 ◽

Vol 21 (18) ◽

pp. 6672

Author(s):

Zora Novakova ◽

Nikola Belousova ◽

Catherine A. Foss ◽

Barbora Havlinova ◽

Marketa Gresova ◽

...

Keyword(s):

Functional Characterization ◽

Xenograft Model ◽

In Vitro Assays ◽

Experimental Therapy ◽

Single Chain ◽

Single Chain Fv ◽

Murine Xenograft Model ◽

Single Target

Prostate-Specific Membrane Antigen (PSMA) is an established biomarker for the imaging and experimental therapy of prostate cancer (PCa), as it is strongly upregulated in high-grade primary, androgen-independent, and metastatic lesions. Here, we report on the development and functional characterization of recombinant single-chain Fv (scFv) and Fab fragments derived from the 5D3 PSMA-specific monoclonal antibody (mAb). These fragments were engineered, heterologously expressed in insect S2 cells, and purified to homogeneity with yields up to 20 mg/L. In vitro assays including ELISA, immunofluorescence and flow cytometry, revealed that the fragments retain the nanomolar affinity and single target specificity of the parent 5D3 antibody. Importantly, using a murine xenograft model of PCa, we verified the suitability of fluorescently labeled fragments for in vivo imaging of PSMA-positive tumors and compared their pharmacokinetics and tissue distribution to the parent mAb. Collectively, our data provide an experimental basis for the further development of 5D3 recombinant fragments for future clinical use.

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Inhibition of NF-κB induces apoptosis of KSHV-infected primary effusion lymphoma cells

Blood ◽

10.1182/blood.v96.7.2537 ◽

2000 ◽

Vol 96 (7) ◽

pp. 2537-2542 ◽

Cited By ~ 229

Author(s):

Shannon A. Keller ◽

Elaine J. Schattner ◽

Ethel Cesarman

Keyword(s):

Primary Effusion Lymphoma ◽

Leukemia Virus ◽

Human Lymphocytes ◽

Kaposi Sarcoma ◽

Irreversible Inhibitor ◽

Mobility Shift ◽

Lymphoma Cells ◽

Barr Virus ◽

Human T Cell ◽

Epstein Barr

Abstract Kaposi sarcoma–associated herpesvirus (KSHV), or human herpervirus 8 (HHV-8), is a γ-herpesvirus that infects human lymphocytes and is associated with primary effusion lymphoma (PEL). Currently, the role of viral infection in the transformation of PEL cells is unknown. One possibility is that KSHV, like the lymphotropic viruses Epstein-Barr virus (EBV) and human T-cell leukemia virus I (HTLV-I), activates the transcription factor NF-κB to promote survival and proliferation of infected lymphocytes. To examine this possibility, we assessed NF-κB activity in KSHV-infected PEL cell lines and primary tumor specimens by electrophoretic mobility shift assay (EMSA). We observed that NF-κB is constitutively activated in all KSHV-infected lymphomas, and consists of 2 predominant complexes, p65/p50 heterodimers and p50/p50 homodimers. Inhibition experiments demonstrated that Bay 11-7082, an irreversible inhibitor of IκBα phosphorylation, completely and specifically abrogated the NF-κB/DNA binding in PEL cells. PEL cells treated with Bay 11 demonstrated down-regulation of the NF-κB inducible cytokine interleukin 6 (IL-6), and apoptosis. These results suggest that NF-κB activity is necessary for survival of KSHV-infected lymphoma cells, and that pharmacologic inhibition of NF-κB may be an effective treatment for PEL.

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Transcriptional coactivation of c-Jun by the KSHV-encoded LANA

Blood ◽

10.1182/blood-2003-05-1538 ◽

2004 ◽

Vol 103 (1) ◽

pp. 222-228 ◽

Cited By ~ 42

Author(s):

Jiabin An ◽

Yiping Sun ◽

Matthew B. Rettig

Keyword(s):

Primary Effusion Lymphoma ◽

Kaposi Sarcoma ◽

Nuclear Antigen ◽

Physical Interaction ◽

Activator Protein 1 ◽

Transcriptional Coactivator ◽

Mobility Shift ◽

Associated Diseases ◽

293 Cells ◽

Electrophoretic Mobility Shift Assays

Abstract The Kaposi sarcoma–associated herpesvirus (KSHV)–encoded latency-associated nuclear antigen (LANA) modulates viral and cellular gene expression, including interleukin 6 (IL-6), a growth factor for KSHV-associated diseases. LANA-driven IL-6 expression is dependent on the activator protein 1 (AP1) response element (RE) within the IL-6 promoter. We show that LANA activates the AP1 RE in a Jun-dependent fashion and that LANA enhances the transcriptional activity of a GAL4-Jun fusion protein. Coimmunoprecipitation studies documented a physical interaction between LANA and c-Jun in transiently transfected 293 cells as well as the KSHV-infected BCBL-1 primary effusion lymphoma (PEL) cell line. Taken together, these data indicate that LANA is a transcriptional coactivator of c-Jun. In addition, electrophoretic mobility shift assays demonstrated that LANA induces binding of a c-Jun-Fos heterodimer to the AP1 RE, but does not itself bind to the AP1 RE. RNA interference experiments confirmed that LANA activates the AP1 RE, stimulates binding of a c-Jun-Fos heterodimer to the AP1 RE, and induces expression of IL-6. These data indicate that LANA is a transcriptional coactivator of c-Jun, a function that may have implications for the pathogenesis of KSHV-associated diseases.

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Triptolide decreases expression of latency-associated nuclear antigen 1 and reduces viral titers in Kaposi's sarcoma-associated and herpesvirus-related primary effusion lymphoma cells

International Journal of Oncology ◽

10.3892/ijo.2016.3353 ◽

2016 ◽

Vol 48 (4) ◽

pp. 1519-1530 ◽

Cited By ~ 11

Author(s):

CONG LONG ◽

WEI GUO ◽

HENG ZHOU ◽

JINGCHAO WANG ◽

HUAN WANG ◽

...

Keyword(s):

Kaposi's Sarcoma ◽

Primary Effusion Lymphoma ◽

Kaposi’S Sarcoma ◽

Nuclear Antigen ◽

Lymphoma Cells ◽

Viral Titers

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Role of IRF4 in IFN-Stimulated Gene Induction and Maintenance of Kaposi Sarcoma–Associated Herpesvirus Latency in Primary Effusion Lymphoma Cells

The Journal of Immunology ◽

10.4049/jimmunol.1202514 ◽

2013 ◽

Vol 191 (3) ◽

pp. 1476-1485 ◽

Cited By ~ 22

Author(s):

Adriana Forero ◽

Patrick S. Moore ◽

Saumendra N. Sarkar

Keyword(s):

Primary Effusion Lymphoma ◽

Kaposi Sarcoma ◽

Gene Induction ◽

Lymphoma Cells

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Human tastin, a proline-rich cytoplasmic protein, associates with the microtubular cytoskeleton

Biochemical Journal ◽

10.1042/bj20011836 ◽

2002 ◽

Vol 364 (3) ◽

pp. 669-677 ◽

Cited By ~ 34

Author(s):

Daita NADANO ◽

Jun NAKAYAMA ◽

Shu-ichi MATSUZAWA ◽

Taka-Aki SATO ◽

Tsukasa MATSUDA ◽

...

Keyword(s):

Mammalian Cells ◽

Molecular Motor ◽

Intracellular Localization ◽

Cytoplasmic Dynein ◽

Binding Activity ◽

In Vitro Assays ◽

Glutathione S Transferase ◽

C Terminus ◽

Microtubular Cytoskeleton

Tastin was originally identified as an accessory protein for trophinin, a cell adhesion molecule that potentially mediates the initial attachment of the human embryo to the uterine epithelium. However, no information regarding tastin's function is available to date. The present study is aimed at understanding the role of tastin in mammalian cells. Hence, we examined the intracellular localization of tastin in human cell lines transfected with an expression vector encoding influenza virus haemagglutinin (HA)-tagged tastin. Ectopically expressed HA—tastin was seen as a pattern resembling the fibres that overlap the microtubular cytoskeleton. When HA—tastin-expressing cells were cultured with nocodazole to disrupt microtubule (MT) polymerization, tastin was dispersed to the entire cytoplasm and an MT sedimentation assay showed tastin in the supernatant; however, tastin was sedimented with polymeric MTs in cell lysates not treated with nocodazole. Sedimentation assays using HA—tastin mutants deleted at the N- or C-terminus revealed MT-binding activity associated with the N-terminal basic region of tastin. A yeast two-hybrid screen for tastin-interacting proteins identified Tctex-1, one of the light chains of cytoplasmic dynein, as a tastin-binding protein. Immunoprecipitation and Western-blot analysis confirmed binding of HA-tagged tastin and FLAG (Asp-Tyr-Lys-Asp-Asp-Asp-Asp-Lys epitope)-tagged Tctex-1 in human cells. Furthermore, in vitro assays have demonstrated the binding between a fusion protein, glutathione S-transferase—Tctex-1, and in vitro translated 35S-labelled tastin. As Tctex-1 is a component of a MT-based molecular motor, these results suggest that tastin plays an important role in mammalian cells by associating with the microtubular cytoskeleton.

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Heparin-Induced Thrombocytopenia and Thrombosis: Therapeutic Strategy Using a Single-Chain Variable Fragment (scFv) Antibody

Blood ◽

10.1182/blood.v120.21.3346.3346 ◽

2012 ◽

Vol 120 (21) ◽

pp. 3346-3346

Author(s):

Jaa Yien New ◽

Jose Perdomo ◽

Xing-Mai Jiang ◽

Beng Chong

Keyword(s):

Monoclonal Antibody ◽

Platelet Aggregation ◽

Heparin Therapy ◽

Human Platelets ◽

Binding Activity ◽

Single Chain Variable Fragment ◽

Single Chain ◽

Heparin Induced Thrombocytopenia ◽

E Coli

Abstract Abstract 3346 Introduction and Aim Heparin-Induced Thrombocytopenia and Thrombosis (HIT) is a life threatening disorder that affects 1–5% of patients receiving heparin therapy. A low platelet count is usually recorded (<150,000 per cubic millimetre) with a decrease of 50% or more from the baseline. The occurrence of HIT is due to the presence of an IgG antibody that recognizes the immune complex formed between Platelet Factor 4 (PF4) and heparin. The antibody/PF4/Heparin complex binds to the FcγRIIa receptor on platelets, leading to platelet activation and thrombotic complications in patients receiving heparin. IV.3 is a murine monoclonal antibody that was raised against the FcγRIIa receptor and has been used as an inhibitor in specificity assays to confirm HIT in patients. We have developed a humanized single-chain variable fragment (scFv) antibody based on the IV.3 monoclonal antibody that binds to the FcγRIIa receptor on platelets and prevents platelet aggregation induced by HIT antibodies. Methods The variable heavy chain (VH) and light chain (VL) of the IV.3 antigen binding fragment (Fab) moiety were amplified using polymerase chain reaction (PCR). These two fragments were then coupled with a linker (Glycine4 and Serine)6. This was followed by introduction of several components including fusion tags (FLAG and c-Myc) at both termini for cloning, detection and purification purposes. The construct was transformed into E. coli (strain-BL21) for protein expression of the scFv. The presence of the protein was detected via immunostaining using anti-FLAG and anti-c-Myc antibodies. The scFv was purified by affinity chromatography and the binding activity was detected using flow cytometry and confocal microscopy. The functional activity was determined using Platelet Aggregation Assay. The scFv was then humanized to minimize potential immunogenicity. Humanization was achieved by introducing specific mutations that rendered the molecule human-like but did not affect binding specificity. The humanized scFv was also expressed in E. coli, purified and tested as before. Results The scFv protein (32kDa) was expressed, purified and confirmed via immunostaining. The created humanized scFv exhibits binding activity against the FcγRIIa on human platelets as determined by flow cytometry and confocal microscopy. In addition, the protein successfully inhibits platelet aggregation at micro molar concentrations in aggregation assays conducted in vitro in the presence of HIT antibodies. Conclusions The humanized scFv was successful in recapitulating the properties of the IV.3 murine monoclonal antibody. It demonstrated binding activity against the FcgRIIa on human platelets and exhibited functional activity by inhibiting platelet activation and aggregation in vitro. This implies that our scFv is able to stop binding of the antibody/PF4/Heparin immune complex to platelets, thus hindering one of the critical initial steps in HIT. The scFv described here may be able to ameliorate the unwanted side effects of heparin therapy and could serve as a potential therapeutic drug for HIT patients. Disclosures: No relevant conflicts of interest to declare.

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