Mimovirus: a Novel Form of Vaccine That Induces Hepatitis B Virus-Specific Cytotoxic T-Lymphocyte Responses In Vivo

ABSTRACT CD8+ cytotoxic T lymphocytes (CTLs) are now recognized as important mediators of immunity against intracellular pathogens, including human immunodeficiency virus and tumors. How to efficiently evoke antigen-specific CTL responses in vivo has become a crucial problem in the development of modern vaccines. Here, we developed a completely novel CTL vaccine—mimovirus, which is a kind of virus-size particulate antigen delivery system. It was formed by the self-assembly of a cationic peptide containing 18 lysines and a CTL-epitope peptide of HBsAg28-39, with a plasmid encoding mouse interleukin-12 (IL-12) through electrostatic interactions. We examined the formation of mimovirus by DNA retardation assay, DNase I protection assay, and transmission electron microscopy and demonstrated that mimovirus could efficiently transfer the plasmid encoding IL-12 into mammalian cells such as P815 cells in vitro. Furthermore, it was proved that mimovirus could induce an HBsAg28-39-specific CTL response in vivo. Considering its effectiveness, flexibility, and defined composition, mimovirus is potentially a novel system for vaccination against intracellular pathogens and tumors.

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Programmable in vitro Co-Encapsidation of Guest Proteins Inside Protein Nanocages

10.26434/chemrxiv.5844393 ◽

2018 ◽

Author(s):

Noor H. Dashti ◽

Rufika S. Abidin ◽

Frank Sainsbury

Keyword(s):

Self Assembly ◽

Mammalian Cells ◽

Resonance Energy Transfer ◽

Resonance Energy ◽

Super Resolution ◽

Cell Engineering ◽

Particle Analysis ◽

Protein Cages

Bioinspired self-sorting and self-assembling systems using engineered versions of natural protein cages have been developed for biocatalysis and therapeutic delivery. The packaging and intracellular delivery of guest proteins is of particular interest for both in vitro and in vivo cell engineering. However, there is a lack of platforms in bionanotechnology that combine programmable guest protein encapsidation with efficient intracellular uptake. We report a minimal peptide anchor for in vivo self-sorting of cargo-linked capsomeres of the Murine polyomavirus (MPyV) major coat protein that enables controlled encapsidation of guest proteins by in vitro self-assembly. Using Förster resonance energy transfer (FRET) we demonstrate the flexibility in this system to support co-encapsidation of multiple proteins. Complementing these ensemble measurements with single particle analysis by super-resolution microscopy shows that the stochastic nature of co-encapsidation is an overriding principle. This has implications for the design and deployment of both native and engineered self-sorting encapsulation systems and for the assembly of infectious virions. Taking advantage of the encoded affinity for sialic acids ubiquitously displayed on the surface of mammalian cells, we demonstrate the ability of self-assembled MPyV virus-like particles to mediate efficient delivery of guest proteins to the cytosol of primary human cells. This platform for programmable co-encapsidation and efficient cytosolic delivery of complementary biomolecules therefore has enormous potential in cell engineering.

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Fusion Hybrid of Dendritic Cells and Engineered Tumor Cells Expressing Interleukin-12 Induces Type 1 Immune Responses against Tumor

Tumori Journal ◽

10.1177/030089160509100614 ◽

2005 ◽

Vol 91 (6) ◽

pp. 531-538 ◽

Cited By ~ 11

Author(s):

Meiqing Shi ◽

Liping Su ◽

Sigou Hao ◽

Xulin Guo ◽

Jim Xiang

Keyword(s):

Cancer Immunotherapy ◽

Tumor Cells ◽

Antitumor Immunity ◽

Cytotoxic T Lymphocyte ◽

Tumor Regression ◽

Interleukin 12 ◽

Myeloma Cells ◽

Th1 Cytokine

Aims and Background Dendritic cell (DC)-tumor fusion hybrid vaccinees that facilitate antigen presentation represent a novel powerful strategy in cancer immunotherapy. Preclinical studies have demonstrated that IL-12 promotes specific antitumor immunity mediated by T cells in several types of tumors. In the present study, we investigated the antitumor immunity derived from vaccination of fusion hybrids between DCs and engineered J558/IL-12 myeloma cells secreting Th1 cytokine IL-12. Methods The expression vector pcDNA-IL-12 was generated and transfected into J558 myeloma cells and then bone marrow-derived DCs were fused with engineered J558/IL-12 cells. The antitumor immunity derived from vaccination of the fusion hybrid DC/J558/IL-12 was evaluated in vitro and in vivo. Results DC/J558/IL-12 cells secreted recombinant IL-12 (1.6 ng/mL), and inoculation of BALB/c mice with DC/J558/IL-12 hybrid induced a Th1 dominant immune response and resulted in tumor regression. Immunization of mice with engineered DC/J558/IL-12 hybrid elicited stronger J558 tumor-specific cytotoxic T lymphocyte (CTL) responses in vitro as well as more potent protective immunity against J558 tumor challenge in vivo than immunization with the mixture of DCs and J558/IL-12, J558/IL-12 and J558, respectively. Furthermore, the antitumor immunity mediated by DC/J558/1L-12 tumor cell vaccination in vivo appeared to be dependent on CD8+ CTL. Conclusions These results demonstrate that the engineered fusion hybrid vaccines that combine Th1 cytokine gene-modified tumor cells with DCs may be an attractive strategy for cancer immunotherapy.

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Phase separation of both a plant virus movement protein and cellular factors support virus-host interactions

10.1101/2021.05.11.443547 ◽

2021 ◽

Author(s):

Shelby L Brown ◽

Jared P. May

Keyword(s):

Phase Separation ◽

Mosaic Virus ◽

Self Assembly ◽

Electrostatic Interactions ◽

Movement Protein ◽

Virus Movement ◽

In Planta ◽

Ribonucleoprotein Complexes

Phase separation concentrates biomolecules, which should benefit RNA viruses that must sequester viral and host factors during an infection. Here, the p26 movement protein from Pea enation mosaic virus 2 (PEMV2) was found to phase separate and partition in nucleoli and G3BP stress granules (SGs) in vivo . Electrostatic interactions drive p26 phase separation as mutation of basic (R/K-G) or acidic (D/E-G) residues either blocked or reduced phase separation, respectively. During infection, p26 must partition inside the nucleolus and interact with fibrillarin (Fib2) as a pre-requisite for systemic trafficking of viral RNAs. Partitioning of p26 in pre-formed Fib2 droplets was dependent on p26 phase separation suggesting that phase separation of viral movement proteins supports nucleolar partitioning and virus movement. Furthermore, viral ribonucleoprotein complexes containing p26, Fib2, and PEMV2 RNA were formed via phase separation in vitro and could provide the basis for self-assembly in planta . Interestingly, both R/K-G and D/E-G p26 mutants failed to support systemic trafficking of a Tobacco mosaic virus (TMV) vector in Nicotiana benthamiana suggesting that p26 phase separation, proper nucleolar partitioning, and systemic movement are intertwined. p26 also partitioned in SGs and G3BP over-expression restricted PEMV2 accumulation >20-fold. Expression of phase separation-deficient G3BP only restricted PEMV2 5-fold, demonstrating that G3BP phase separation is critical for maximum antiviral activity.

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Gene-Activated Matrix with Self-Assembly Anionic Nano-Device Containing Plasmid DNAs for Rat Cranial Bone Augmentation

Materials ◽

10.3390/ma14227097 ◽

2021 ◽

Vol 14 (22) ◽

pp. 7097

Author(s):

Masahito Hara ◽

Yoshinori Sumita ◽

Yukinobu Kodama ◽

Mayumi Iwatake ◽

Hideyuki Yamamoto ◽

...

Keyword(s):

Self Assembly ◽

Electrostatic Interactions ◽

Cranial Bone ◽

Bone Augmentation ◽

Mesenchymal Progenitor Cells ◽

Anionic Polymer ◽

Morphogenetic Protein ◽

Low Cytotoxicity

We have developed nanoballs, a biocompatible self-assembly nano-vector based on electrostatic interactions that arrange anionic macromolecules to polymeric nanomaterials to create nucleic acid carriers. Nanoballs exhibit low cytotoxicity and high transfection efficiently in vivo. This study investigated whether a gene-activated matrix (GAM) composed of nanoballs containing plasmid (p) DNAs encoding bone morphogenetic protein 4 (pBMP4) could promote bone augmentation with a small amount of DNA compared to that composed of naked pDNAs. We prepared nanoballs (BMP4-nanoballs) constructed with pBMP4 and dendrigraft poly-L-lysine (DGL, a cationic polymer) coated by γ-polyglutamic acid (γ-PGA; an anionic polymer), and determined their biological functions in vitro and in vivo. Next, GAMs were manufactured by mixing nanoballs with 2% atelocollagen and β-tricalcium phosphate (β-TCP) granules and lyophilizing them for bone augmentation. The GAMs were then transplanted to rat cranial bone surfaces under the periosteum. From the initial stage, infiltrated macrophages and mesenchymal progenitor cells took up the nanoballs, and their anti-inflammatory and osteoblastic differentiations were promoted over time. Subsequently, bone augmentation was clearly recognized for up to 8 weeks in transplanted GAMs containing BMP4-nanoballs. Notably, only 1 μg of BMP4-nanoballs induced a sufficient volume of new bone, while 1000 μg of naked pDNAs were required to induce the same level of bone augmentation. These data suggest that applying this anionic vector to the appropriate matrices can facilitate GAM-based bone engineering.

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Anthrax Toxin-Mediated Delivery In Vivo and In Vitro of a Cytotoxic T-Lymphocyte Epitope from Ovalbumin

Infection and Immunity ◽

10.1128/iai.66.2.615-619.1998 ◽

1998 ◽

Vol 66 (2) ◽

pp. 615-619 ◽

Cited By ~ 40

Author(s):

Jimmy D. Ballard ◽

Amy M. Doling ◽

Kathryn Beauregard ◽

R. John Collier ◽

Michael N. Starnbach

Keyword(s):

Fusion Protein ◽

T Lymphocyte ◽

Self Assembly ◽

Protective Antigen ◽

Cytotoxic T Lymphocyte ◽

Ex Vivo ◽

Lethal Factor ◽

Anthrax Toxin

ABSTRACT We reported earlier that a nontoxic form of anthrax toxin was capable of delivering a cytotoxic T-lymphocyte (CTL) epitope in vivo, such that a specific CTL response was primed against the epitope. The epitope, of bacterial origin, was fused to an N-terminal fragment (LFn) from the lethal-factor component of the toxin, and the fusion protein was injected, together with the protective antigen (PA) component, into BALB/c mice. Here we report that PA plus LFn is capable of delivering a different epitope—OVA257–264 from ovalbumin. Delivery was accomplished in a different mouse haplotype,H-2Kb and occurred in vitro as well as in vivo. An OVA257–264-specific CTL clone, GA-4, recognized EL-4 cells treated in vitro with PA plus as little as 30 fmol of the LFn-OVA257–264 fusion protein. PA mutants attenuated in toxin self-assembly or translocation were inactive, implying that the role of PA in epitope delivery is the same as that in toxin action. Also, we showed that OVA257–264-specific CTL could be induced to proliferate by incubation with splenocytes treated with PA plus LFn-OVA257–264. These findings imply that PA-LFn may serve as a general delivery vehicle for CTL epitopes in vivo and as a safe, efficient tool for the ex vivo expansion of patient-derived CTL for use in adoptive immunotherapy.

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Enhancement of Innate Immunity againstMycobacterium avium Infection by Immunostimulatory DNA Is Mediated by Indoleamine 2,3-Dioxygenase

Infection and Immunity ◽

10.1128/iai.69.10.6156-6164.2001 ◽

2001 ◽

Vol 69 (10) ◽

pp. 6156-6164 ◽

Cited By ~ 49

Author(s):

Tomoko Hayashi ◽

Savita P. Rao ◽

Kenji Takabayashi ◽

John H. Van Uden ◽

Richard S. Kornbluth ◽

...

Keyword(s):

Innate Immunity ◽

Cytotoxic T Lymphocyte ◽

Interleukin 12 ◽

Necrosis Factor Alpha ◽

Indoleamine 2,3 Dioxygenase ◽

Mouse Macrophages ◽

Avium Infection ◽

Human And Mouse

ABSTRACT Bacterial DNA and its synthetic immunostimulatory oligodeoxynucleotide analogs (ISS-ODN) activate innate immunity and promote Th1 and cytotoxic T-lymphocyte immune responses. Based on these activities, we investigated whether ISS-ODN could modify the course ofMycobacterium avium infection. M. aviumgrowth in vitro was significantly inhibited by ISS-ODN treatment of human and mouse macrophages, and M. avium growth in vivo was similarly inhibited in C57BL/6 mice treated with ISS-ODN. This protective effect of ISS-ODN was largely independent of tumor necrosis factor alpha (TNF-α), interleukin 12 (IL-12), nitric oxide, NADPH oxidase, alpha/beta interferon (IFN-α/β), and IFN-γ. In contrast, we found that the induction of indoleamine 2,3-dioxygenase (IDO) was required for the antimycobacterial effect of ISS-ODN. To evaluate the potential for synergism between ISS-ODN and other antimycobacterial agents, treatment with a combination of ISS-ODN and clarithromycin (CLA) was tested in vitro and in vivo. ISS-ODN significantly enhanced the therapeutic effect of CLA in both human and mouse macrophages and in C57BL/6 mice. This study newly identifies IDO as being involved in the antimicrobial activity of ISS-ODN and suggests the usefulness of ISS-ODN when used in combination with conventional chemotherapy for microbial infections.

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Programmable in vitro Co-Encapsidation of Guest Proteins Inside Protein Nanocages

10.26434/chemrxiv.5844393.v1 ◽

2018 ◽

Author(s):

Noor H. Dashti ◽

Rufika S. Abidin ◽

Frank Sainsbury

Keyword(s):

Self Assembly ◽

Mammalian Cells ◽

Resonance Energy Transfer ◽

Resonance Energy ◽

Super Resolution ◽

Cell Engineering ◽

Particle Analysis ◽

Protein Cages

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Vaccines With Interleukin-12–Transduced Acute Myeloid Leukemia Cells Elicit Very Potent Therapeutic and Long-Lasting Protective Immunity

Blood ◽

10.1182/blood.v94.12.4263 ◽

1999 ◽

Vol 94 (12) ◽

pp. 4263-4273 ◽

Cited By ~ 50

Author(s):

Kyriaki Dunussi-Joannopoulos ◽

Kathlene Runyon ◽

Jamie Erickson ◽

Robert G. Schaub ◽

Robert G. Hawley ◽

...

Keyword(s):

T Cells ◽

Acute Myeloid Leukemia ◽

Myeloid Leukemia ◽

Residual Disease ◽

Cytotoxic T Lymphocyte ◽

Interleukin 12 ◽

Ifn Γ ◽

Acute Myeloid

Abstract Interleukin-12 (IL-12) is a heterodimeric cytokine mediating a dynamic interplay between T cells and antigen-presenting cells (APCs). Preclinical studies have demonstrated that recombinant murine IL-12 (rmIL-12) promotes specific antitumor immunity mediated by T cells in several types of tumors. However, the in vivo antitumor properties of IL-12 in acute myeloid leukemia (AML) have not been previously reported. We show here in a murine AML model that systemic administration of rmIL-12 significantly delays tumor growth but is incapable of rescuing mice from lethal leukemia. In contrast, AML cells genetically modified to express IL-12 (IL12-AML) using murine stem cell virus (MSCV) p40 + p35 elicit very potent antileukemic activity. Vaccines with lethally irradiated IL12-AML cells protect naive mice against challenge with wild-type AML cells and, more importantly, can cure mice bearing a considerable leukemic burden. Immunized mice show no signs of systemic IL-12 toxicity and their spleen histology is comparable with naive mice spleen. In vivo depletion of IL-12, interferon-γ (IFN-γ), or CD8+ T cells after injections with live IL12-AML cells abrogates completely the antileukemia immune responses. Studies on the in vitro effects of IFN-γ on AML cells demonstrate enhanced expression of major histocompatibility complex (MHC) and accessory molecules and induction of the costimulatory molecules B7.1 and B7.2, but no significant direct antiproliferative effect. 51Cr release assays show that rejection of live IL12-AML cells supports the development of long-lasting leukemia-specific cytotoxic T lymphocyte (CTL) activity. In conclusion, our results demonstrate that IL12-AML vaccination is a safe and potent immunotherapeutic approach that has a great potential to eliminate minimal residual disease in patients with AML.

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Leucine 232 and hydrophobic residues at the ribosomal P stalk binding site are critical for biological activity of ricin

Bioscience Reports ◽

10.1042/bsr20192022 ◽

2019 ◽

Vol 39 (10) ◽

Cited By ~ 2

Author(s):

Yijun Zhou ◽

Xiao-Ping Li ◽

Jennifer N. Kahn ◽

John E. McLaughlin ◽

Nilgun E. Tumer

Keyword(s):

Biological Activity ◽

Mammalian Cells ◽

Electrostatic Interactions ◽

Active Site Residues ◽

Hydrophobic Pocket ◽

Hydrophobic Residues ◽

A Chain ◽

Ribosomal P

Abstract Ricin interacts with the ribosomal P stalk to cleave a conserved adenine from the α-sarcin/ricin loop (SRL) of the rRNA. Ricin toxin A chain (RTA) uses Arg235 as the most critical arginine for binding to the P stalk through electrostatic interactions to facilitate depurination. Structural analysis showed that a P2 peptide binds to a hydrophobic pocket on RTA and the last two residues form hydrogen bonds with Arg235. The importance of hydrophobic residues relative to Arg235 in the interaction with the P stalk in vivo and on the toxicity of RTA is not known. Here, we mutated residues in the hydrophobic pocket to analyze their contribution to toxicity and depurination activity in yeast and in mammalian cells. We found that Leu232, Tyr183 and Phe240 contribute cumulatively to toxicity, with Leu232 being the most significant. A quadruple mutant, Y183A/L232A/R235A/F240A, which combined mutations in critical hydrophobic residues with R235A completely abolished the activity of RTA, indicating that Arg235 and hydrophobic residues are required for full biological activity. Y183A and F240A mutants had reduced activity on RNA, but higher activity on ribosomes compared with R235A in vitro, suggesting that they could partially regain activity upon interaction with ribosomes. These results expand the region of interaction between RTA and the P stalk critical for cellular activity to include the hydrophobic pocket and provide the first evidence that interaction of P stalk with the hydrophobic pocket promotes a conformational rearrangement of RTA to correctly position the active site residues for catalytic attack on the SRL.

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Heparin modifies the immunogenicity of positively charged proteins

Blood ◽

10.1182/blood-2010-06-292938 ◽

2010 ◽

Vol 116 (26) ◽

pp. 6046-6053 ◽

Cited By ~ 34

Author(s):

Shalini L. Chudasama ◽

Benjamin Espinasse ◽

Fred Hwang ◽

Rui Qi ◽

Manali Joglekar ◽

...

Keyword(s):

Electrostatic Interactions ◽

Cell Activation ◽

High Titer ◽

Interleukin 12 ◽

Dendritic Cell Activation ◽

Platelet Factor ◽

Heparin Induced Thrombocytopenia ◽

Positively Charged

Abstract The immune response in heparin-induced thrombocytopenia is initiated by and directed to large multimolecular complexes of platelet factor 4 (PF4) and heparin (H). We have previously shown that PF4:H multimolecular complexes assemble through electrostatic interactions and, once formed, are highly immunogenic in vivo. Based on these observations, we hypothesized that other positively charged proteins would exhibit similar biologic interactions with H. To test this hypothesis, we selected 2 unrelated positively charged proteins, protamine (PRT) and lysozyme, and studied H-dependent interactions using in vitro and in vivo techniques. Our studies indicate that PRT/H and lysozyme/H, like PF4/H, show H-dependent binding over a range of H concentrations and that formation of complexes occurs at distinct stoichiometric ratios. We show that protein/H complexes are capable of eliciting high-titer antigen-specific antibodies in a murine immunization model and that PRT/H antibodies occur in patients undergoing cardiopulmonary bypass surgery. Finally, our studies indicate that protein/H complexes, but not uncomplexed protein, directly activate dendritic cells in vitro leading to interleukin-12 release. Taken together, these studies indicate that H significantly alters the biophysical and biologic properties of positively charged compounds through formation of multimolecular complexes that lead to dendritic cell activation and trigger immune responses in vivo.

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