Publications by authors named "Nicole F Steinmetz"

178 Publications

Cowpea mosaic virus stimulates antitumor immunity through recognition by multiple MYD88-dependent toll-like receptors.

Biomaterials 2021 May 25;275:120914. Epub 2021 May 25.

Department of Microbiology and Immunology, Geisel School of Medicine, Dartmouth College, Hanover, NH, 03755, United States; Norris Cotton Cancer Center, Geisel School of Medicine, Dartmouth Hitchcock Medical System, Lebanon, NH, 03756, United States. Electronic address:

Cowpea mosaic virus (CPMV), a non-enveloped plant virus, and empty CPMV (eCPMV), a virus-like particle (VLP) composed of CPMV capsid without nucleic acids, are potent in situ cancer vaccines when administered intratumorally (I.T.). However, it is unclear how immune cells recognize these nanoparticles and why they are immunogenic, which was investigated in this study. CPMV generated stronger selective induction of cytokines and chemokines in naïve mouse splenocytes and exhibited more potent anti-tumor efficacy than eCPMV. MyD88 is required for both CPMV- and eCPMV-elicited immune responses. Screening with human embryonic kidney (HEK)-293 cell toll-like receptor (TLR) reporter assays along with experiments in corresponding TLR-/- mice indicated CPMV and eCPMV capsids are recognized by MyD88-dependent TLR2 and TLR4. CPMV, but not eCPMV, is additionally recognized by TLR7. Secretion of type I interferons (IFNs), which requires the interaction between TLR7 and encapsulated single-stranded RNAs (ssRNAs), is critical to CPMV's better efficacy. The same recognition mechanisms are also functional in human peripheral blood mononuclear cells (PBMCs). Overall, these findings link CPMV immunotherapy efficacy with molecular recognition, provide rationale for how to develop more potent viral particles, accentuate the value of multi-TLR agonists as in situ cancer vaccines, and highlight the functional importance of type I IFNs for in situ vaccination.
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http://dx.doi.org/10.1016/j.biomaterials.2021.120914DOI Listing
May 2021

Designing S100A9-Targeted Plant Virus Nanoparticles to Target Deep Vein Thrombosis.

Biomacromolecules 2021 Jun 1;22(6):2582-2594. Epub 2021 Jun 1.

Department of NanoEngineering, University of California, San Diego, La Jolla, California 92093, United States.

Thromboembolic conditions are a leading cause of death worldwide, and deep vein thrombosis (DVT), or occlusive venous clot formation, is a critical and rising problem that contributes to damage of vital organs, long-term complications, and life-threatening conditions such as pulmonary embolism. Early diagnosis and treatment are correlated to better prognosis. However, current technologies in these areas, such as ultrasonography for diagnostics and anticoagulants for treatment, are limited in terms of their accuracy and therapeutic windows. In this work, we investigated targeting myeloid related protein 14 (MRP-14, also known as S100A9) using plant virus-based nanoparticle carriers as a means to achieve tissue specificity aiding prognosis and therapeutic intervention. We used a combinatorial peptide library screen to identify peptide ligands that bind MRP-14. Candidates were selected and formulated as nanoparticles by using cowpea mosaic virus (CPMV) and tobacco mosaic virus (TMV). Intravascular delivery of our MRP-14-targeted nanoparticles in a murine model of DVT resulted in enhanced accumulation in the thrombi and reduced thrombus size, suggesting application of nanoparticles for molecular targeting of MRP-14 could be a promising direction for improving DVT diagnostics, therapeutics, and therefore prognosis.
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http://dx.doi.org/10.1021/acs.biomac.1c00303DOI Listing
June 2021

Active Microneedle Administration of Plant Virus Nanoparticles for Cancer Vaccination Improves Immunotherapeutic Efficacy.

ACS Appl Nano Mater 2020 Aug 7;3(8):8037-8051. Epub 2020 Aug 7.

Department of Radiology, UC San Diego Health, University of California, San Diego, La Jolla California 92093, United States.

The solid tumor microenvironment (TME) poses a significant structural and biochemical barrier to immunotherapeutic agents. To address the limitations of tumor penetration and distribution, and to enhance antitumor efficacy of immunotherapeutics, we present here an autonomous active microneedle (MN) system for the direct intratumoral (IT) delivery of a potent immunoadjuvant, cowpea mosaic virus nanoparticles (CPMV) . In this active delivery system, magnesium (Mg) microparticles embedded into active MNs react with the interstitial fluid in the TME, generating a propulsive force to drive the nanoparticle payload into the tumor. Active delivery of CPMV payload into B16F10 melanomas demonstrated substantially more pronounced tumor regression and prolonged survival of tumor-bearing mice compared to that of passive MNs and conventional needle injection. Active MN administration of CPMV also enhanced local innate and systemic adaptive antitumor immunity. Our approach represents an elaboration of conventional CPMV vaccination, highlighting substantial immune-mediated antitumor effects and improved therapeutic efficacy that can be achieved through an active and autonomous delivery system-mediated CPMV vaccination.
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http://dx.doi.org/10.1021/acsanm.0c01506DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8101548PMC
August 2020

Unleashing the potential of cell membrane-based nanoparticles for COVID-19 treatment and vaccination.

Expert Opin Drug Deliv 2021 Jun 6:1-20. Epub 2021 Jun 6.

Faculty of Pharmacy of the University of Coimbra, University of Coimbra, Coimbra, Portugal.

: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a particular coronavirus strain responsible for the coronavirus disease 2019 (COVID-19), accounting for more than 3.1 million deaths worldwide. Several health-related strategies have been successfully developed to contain the rapidly-spreading virus across the globe, toward reduction of both disease burden and infection rates. Particularly, attention has been focused on either the development of novel drugs and vaccines, or by adapting already-existing drugs for COVID-19 treatment, mobilizing huge efforts to block disease progression and to overcome the shortage of effective measures available at this point.: This perspective covers the breakthrough of multifunctional biomimetic cell membrane-based nanoparticles as next-generation nanosystems for cutting-edge COVID-19 therapeutics and vaccination, specifically cell membrane-derived nanovesicles and cell membrane-coated nanoparticles, both tailorable cell membrane-based nanosystems enriched with the surface repertoire of native cell membranes, toward maximized biointerfacing, immune evasion, cell targeting and cell-mimicking properties.: Nano-based approaches have received widespread interest regarding enhanced antigen delivery, prolonged blood circulation half-life and controlled release of drugs. Cell membrane-based nanoparticles comprise interesting antiviral multifunctional nanoplatforms for blocking SARS-CoV-2 binding to host cells, reducing inflammation through cytokine neutralization and improving drug delivery toward COVID-19 treatment.
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http://dx.doi.org/10.1080/17425247.2021.1922387DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8182831PMC
June 2021

Bluetongue Virus Particles as Nanoreactors for Enzyme Delivery and Cancer Therapy.

Mol Pharm 2021 03 10;18(3):1150-1156. Epub 2021 Feb 10.

Department of Biomedical Engineering, Case Western Reserve University, Cleveland, Ohio 44106, United States.

The side effects of chemotherapy can be reduced by targeting tumor cells with an enzyme (or the corresponding gene) that converts a nontoxic prodrug into a toxic drug inside the tumor cells, also killing the surrounding tumor cells via the bystander effect. Viruses are the most efficient gene delivery vehicles because they have evolved to transfer their own nucleic acids into cells, but their efficiency must be balanced against the risks of infection, the immunogenicity of nucleic acids, and the potential for genomic integration. We therefore tested the effectiveness of genome-free virus-like particles (VLPs) for the delivery of Herpes simplex virus 1 thymidine kinase (HSV1-TK), the most common enzyme used in prodrug conversion therapy. HSV1-TK is typically delivered as a gene, but in the context of VLPs, it must be delivered as a protein. We constructed VLPs and smaller core-like particles (CLPs) based on Bluetongue virus, with HSV1-TK fused to the inner capsid protein VP3. TK-CLPs and TK-VLPs could be produced in large quantities in plants. The TK-VLPs killed human glioblastoma cells efficiently in the presence of ganciclovir, with an IC value of 14.8 μM. Conversely, CLPs were ineffective because they remained trapped in the endosomal compartment, in common with many synthetic nanoparticles. VLPs are advantageous because they can escape from endosomes and therefore allow HSV1-TK to access the cytosolic adenosine triphosphate (ATP) required for the phosphorylation of ganciclovir. The VLP delivery strategy of TK protein therefore offers a promising new modality for the treatment of cancer with systemic prodrugs such as ganciclovir.
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http://dx.doi.org/10.1021/acs.molpharmaceut.0c01053DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8086382PMC
March 2021

Remission-Stage Ovarian Cancer Cell Vaccine with Cowpea Mosaic Virus Adjuvant Prevents Tumor Growth.

Cancers (Basel) 2021 Feb 5;13(4). Epub 2021 Feb 5.

Department of Microbiology and Immunology, Geisel School of Medicine, Dartmouth College, Lebanon, NH 03756, USA.

Ovarian cancer is the deadliest gynecological malignancy. Though most patients enter remission following initial interventions, relapse is common and often fatal. Accordingly, there is a substantial need for ovarian cancer therapies that prevent relapse. Following remission generated by surgical debulking and chemotherapy, but prior to relapse, resected and inactivated tumor tissue could be used as a personalized vaccine antigen source. The patient's own tumor contains relevant antigens and, when combined with the appropriate adjuvant, could generate systemic antitumor immunity to prevent relapse. Here, we model this process in mice to investigate the optimal tumor preparation and vaccine adjuvant. Cowpea mosaic virus (CPMV) has shown remarkable efficacy as an immunostimulatory cancer therapy in ovarian cancer mouse models, so we use CPMV as an adjuvant in a prophylactic vaccine against a murine ovarian cancer model. Compared to its codelivery with tumor antigens prepared in three other ways, we show that CPMV co-delivered with irradiated ovarian cancer cells constitutes an effective prophylactic vaccine against a syngeneic model of ovarian cancer in C57BL/6J mice. Following two vaccinations, 72% of vaccinated mice reject tumor challenges, and all those mice survived subsequent rechallenges, demonstrating immunologic memory formation. This study supports remission-stage vaccines using irradiated patient tumor tissue as a promising option for treating ovarian cancer, and validates CPMV as an antitumor vaccine adjuvant for that purpose.
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http://dx.doi.org/10.3390/cancers13040627DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7915664PMC
February 2021

The pharmacology of plant virus nanoparticles.

Virology 2021 Apr 28;556:39-61. Epub 2021 Jan 28.

Department of NanoEngineering, University of California-San Diego, La Jolla, CA, 92039, United States; Department of Bioengineering, Department of Radiology, Center for NanoImmunoEngineering, Moores Cancer Center, Institute for Materials Discovery and Design, University of California-San Diego, La Jolla, CA, 92039, United States. Electronic address:

The application of nanoparticles for medical purposes has made enormous strides in providing new solutions to health problems. The observation that plant virus-based nanoparticles (VNPs) can be repurposed and engineered as smart bio-vehicles for targeted drug delivery and imaging has launched extensive research for improving the therapeutic and diagnostic management of various diseases. There is evidence that VNPs are promising high value nanocarriers with potential for translational development. This is mainly due to their unique features, encompassing structural uniformity, ease of manufacture and functionalization by means of expression, chemical biology and self-assembly. While the development pipeline is moving rapidly, with many reports focusing on engineering and manufacturing aspects to tailor the properties and efficacy of VNPs, fewer studies have focused on gaining insights into the nanotoxicity of this novel platform nanotechnology. Herein, we discuss the pharmacology of VNPs as a function of formulation and route of administration. VNPs are reviewed in the context of their application as therapeutic adjuvants or nanocarrier excipients to initiate, enhance, attenuate or impede the formulation's toxicity. The summary of the data however also underlines the need for meticulous VNP structure-nanotoxicity studies to improve our understanding of their in vivo fates and pharmacological profiles to pave the way for translation of VNP-based formulations into the clinical setting.
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http://dx.doi.org/10.1016/j.virol.2021.01.012DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7974633PMC
April 2021

Virus-Like Particles as Positive Controls for COVID-19 RT-LAMP Diagnostic Assays.

Biomacromolecules 2021 03 4;22(3):1231-1243. Epub 2021 Feb 4.

Reverse transcription loop-mediated isothermal amplification (RT-LAMP) is a rapid and inexpensive isothermal alternative to the current gold standard reverse transcription quantitative polymerase chain reaction (RT-qPCR) for the detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). However, unlike RT-qPCR, there are no consensus detection regions or optimal RT-LAMP methods, and most protocols do not include internal controls to ensure reliability. Naked RNAs, plasmids, or even RNA from infectious COVID-19 patients have been used as external positive controls for RT-LAMP assays, but such reagents lack the stability required for full-process control. To overcome the lack of proper internal and external positive controls and the instability of the detection RNA, we developed virus-like particles (VLPs) using bacteriophage Qβ and plant virus cowpea chlorotic mottle virus (CCMV) for the encapsidation of target RNA, namely a so-called SARS-CoV-2 LAMP detection module (SLDM). The target RNA is a truncated segment of the SARS-CoV-2 nucleocapsid (N) gene and human RNase P gene (internal control) as positive controls for RT-qPCR and RT-LAMP. Target RNAs stably encapsidated in Qβ and CCMV VLPs were previously shown to function as full-process controls in RT-qPCR assays, and here we show that SLDMs can fulfill the same function for RT-LAMP and swab-to-test (direct RT-LAMP with heat lysis) assays. The SLDM was validated in a clinical setting, highlighting the promise of VLPs as positive controls for molecular assays.
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http://dx.doi.org/10.1021/acs.biomac.0c01727DOI Listing
March 2021

A Scalable Manufacturing Approach to Single Dose Vaccination against HPV.

Vaccines (Basel) 2021 Jan 19;9(1). Epub 2021 Jan 19.

Department of NanoEngineering, University of California San Diego, La Jolla, CA 92093, USA.

Human papillomavirus (HPV) is a globally prevalent sexually-transmitted pathogen, responsible for most cases of cervical cancer. HPV vaccination rates remain suboptimal, partly due to the need for multiple doses, leading to a lack of compliance and incomplete protection. To address the drawbacks of current HPV vaccines, we used a scalable manufacturing process to prepare implantable polymer-protein blends for single-administration with sustained delivery. Peptide epitopes from HPV16 capsid protein L2 were conjugated to the virus-like particles derived from bacteriophage Qβ, to enhance their immunogenicity. The HPV-Qβ particles were then encapsulated into poly(lactic-co-glycolic acid) (PLGA) implants, using a benchtop melt-processing system. The implants facilitated the slow and sustained release of HPV-Qβ particles without the loss of nanoparticle integrity, during high temperature melt processing. Mice vaccinated with the implants generated IgG titers comparable to the traditional soluble injections and achieved protection in a pseudovirus neutralization assay. HPV-Qβ implants offer a new vaccination platform; because the melt-processing is so versatile, the technology offers the opportunity for massive upscale into any geometric form factor. Notably, microneedle patches would allow for self-administration in the absence of a healthcare professional, within the developing world. The Qβ technology is highly adaptable, allowing the production of vaccine candidates and their delivery devices for multiple strains or types of viruses.
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http://dx.doi.org/10.3390/vaccines9010066DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7835769PMC
January 2021

Cowpea Mosaic Virus Nanoparticle Enhancement of Hypofractionated Radiation in a B16 Murine Melanoma Model.

Front Oncol 2020 16;10:594614. Epub 2020 Dec 16.

Geisel School of Medicine, Dartmouth College, Hanover, NH, United States.

Introduction: Virus and virus-like nanoparticles (VNPs) have been used for a variety of preclinical treatments, including anti-cancer vaccination. The Cowpea mosaic virus (CPMV) is a VNP that has shown the ability to stimulate an anti-cancer immune response. The hypothesis of this study is two-fold: that intratumoral CPMV enhances the immunogenetic and cytotoxic response of hypofractionated radiation (15 Gy or 3 x 8 Gy), and that the effect differs between fraction regimens in the murine B16 flank melanoma model.

Methods: CPMV nanoparticles were delivered intratumorally, 100 μg/tumor to B16 murine melanoma flank tumors alone, and in combination with either 15 Gy or 3 x 8 Gy (3 consecutive days). Tumors were assessed for immune and cytotoxic gene and protein expression, and cytotoxic T cell infiltration 4 days post treatment. Treatment based tumor control was assessed by a 3-fold tumor growth assay.

Results: Both CPMV and radiation alone demonstrated the activation of a number of important immune and cytotoxic genes including natural killer cell and T cell mediated cytotoxicity pathways. However, the combination treatment activated greater expression than either treatment alone. CPMV combined with a single dose of 15 Gy demonstrated greater immune and cytotoxic gene expression, protein expression, CD8+ T cell infiltration activity, and greater tumor growth delay compared to 3 x 8 Gy with CPMV.

Conclusion: CPMV presents a unique and promising hypofractionated radiation adjuvant that leads to increased anti-tumor cytotoxic and immune signaling, especially with respect to the immune mediated cytotoxicity, immune signaling, and toll-like receptor signaling pathways. This improvement was greater with a single dose than with a fractionated dose.
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http://dx.doi.org/10.3389/fonc.2020.594614DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7773968PMC
December 2020

Biomimetic Virus-Like Particles as Severe Acute Respiratory Syndrome Coronavirus 2 Diagnostic Tools.

ACS Nano 2021 01 25;15(1):1259-1272. Epub 2020 Nov 25.

Coronavirus disease 2019 (COVID-19) is a highly transmissible disease that has affected more than 90% of the countries worldwide. At least 17 million individuals have been infected, and some countries are still battling first or second waves of the pandemic. Nucleic acid tests, especially reverse transcription polymerase chain reaction (RT-PCR), have become the workhorse for early detection of COVID-19 infection. Positive controls for the molecular assays have been developed to validate each test and to provide high accuracy. However, most available positive controls require cold-chain distribution and cannot serve as full-process control. To overcome these shortcomings, we report the production of biomimetic virus-like particles (VLPs) as SARS-CoV-2 positive controls. A SARS-CoV-2 detection module for RT-PCR was encapsidated into VLPs from a bacteriophage and a plant virus. The chimeric VLPs were obtained either by reconstitution and coexpression of the target detection module and coat proteins or by assembly of purified detection module RNA sequences and coat proteins. These VLP-based positive controls mimic SARS-CoV-2 packaged ribonucleic acid (RNA) while being noninfectious. Most importantly, we demonstrated that the positive controls are scalable, stable, and can serve broadly as controls, from RNA extraction to PCR in clinical settings.
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http://dx.doi.org/10.1021/acsnano.0c08430DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7724985PMC
January 2021

Cisplatin Prodrug-Loaded Nanoparticles Based on Physalis Mottle Virus for Cancer Therapy.

Mol Pharm 2020 12 13;17(12):4629-4636. Epub 2020 Nov 13.

Department of NanoEngineering, University of California-San Diego, La Jolla, California 92093, United States.

Nanoparticle-based prodrugs offer an effective strategy to improve the safety and delivery of small-molecule therapeutics while reducing the risk of drug resistance. Here, we conjugated a maleimide-functionalized cisplatin prodrug containing Pt(IV) to the internal and/or external surface of virus-like particles (VLPs) derived from Physalis mottle virus (PhMV) to develop a pH-sensitive drug delivery system. The internally loaded and PEGylated VLPs (Pt-PhMVCy5.5-PEG) were taken up efficiently by cancer cells where they released platinum, presumably as a reduced, DNA-reactive Pt(II) complex, rapidly under acidic conditions (>80% in 30 h). The efficacy of the VLP-based drug delivery system was demonstrated against a panel of cancer cell lines, including cell lines resistant to platinum therapy. Furthermore, Pt-PhMVCy5.5-PEG successfully inhibited the growth of xenograft MDA-MB-231 breast tumors and significantly prolonged the survival of mice compared to free cisplatin and cisplatin-maleimide. Pt-PhMVCy5.5-PEG therefore appears promising as a prodrug to overcome the limitations of conventional platinum-based drugs for cancer therapy.
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http://dx.doi.org/10.1021/acs.molpharmaceut.0c00834DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8087187PMC
December 2020

Charge Calibration Standard for Atomic Force Microscope Tips in Liquids.

Langmuir 2020 11 6;36(45):13621-13632. Epub 2020 Nov 6.

Department of Physics, Yeshiva University, Manhattan, New York 10033, United States.

An electric charge standard with nanoscale resolution is created using the known charge distribution of a single tobacco mosaic virus coat protein combined with the known packing of these proteins in the virus capsid. This advances the ability to measure charge on nanometric samples. Experimental atomic force microscope (AFM) force-distance curves are collected under aqueous conditions with controlled pH and ion concentration. A mathematical model that considers a polarizable dielectric tip immersed in an electrolyte is used to obtain charge density from the AFM measurements. Interactions between the tip and the sample are modeled using theory that includes monopolar electrostatic interactions, dipolar interactions, screening from both the dielectric nature of ambient water and solvated ions as described by the linear Poisson-Boltzmann equation, and hard-core repulsion. It is found that the tip charge density changes on a timescale of hours requiring recalibration of the tip for experiments lasting more than an hour. As an example of how a charge-calibrated tip may be used, the surface charge densities on 20 individual carboxylate-modified polystyrene (PS) beads are measured. The average of these AFM-measured bead charge densities is compared with the value obtained from conventional titration combined with electron microscopy. The two values are found to agree within 20%. While the comparison demonstrates similarity of the two charge measurements, hypotheses are put forward as to why the two techniques might be expected not to provide identical mean charge densities. The considerations used to build these hypotheses thus underscore the relevance of the method performed here if charge information is required on individual nanoparticles.
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http://dx.doi.org/10.1021/acs.langmuir.0c02455DOI Listing
November 2020

COVID-19 Vaccine Frontrunners and Their Nanotechnology Design.

ACS Nano 2020 10 9;14(10):12522-12537. Epub 2020 Oct 9.

Department of Bioengineering, University of California San Diego, La Jolla, California 92093, United States.

Humanity is experiencing a catastrophic pandemic. SARS-CoV-2 has spread globally to cause significant morbidity and mortality, and there still remain unknowns about the biology and pathology of the virus. Even with testing, tracing, and social distancing, many countries are struggling to contain SARS-CoV-2. COVID-19 will only be suppressible when herd immunity develops, either because of an effective vaccine or if the population has been infected and is resistant to reinfection. There is virtually no chance of a return to pre-COVID-19 societal behavior until there is an effective vaccine. Concerted efforts by physicians, academic laboratories, and companies around the world have improved detection and treatment and made promising early steps, developing many vaccine candidates at a pace that has been unmatched for prior diseases. As of August 11, 2020, 28 of these companies have advanced into clinical trials with Moderna, CanSino, the University of Oxford, BioNTech, Sinovac, Sinopharm, Anhui Zhifei Longcom, Inovio, Novavax, Vaxine, Zydus Cadila, Institute of Medical Biology, and the Gamaleya Research Institute having moved beyond their initial safety and immunogenicity studies. This review analyzes these frontrunners in the vaccine development space and delves into their posted results while highlighting the role of the nanotechnologies applied by all the vaccine developers.
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http://dx.doi.org/10.1021/acsnano.0c07197DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7553041PMC
October 2020

Plant Viruses and Bacteriophage-Based Reagents for Diagnosis and Therapy.

Annu Rev Virol 2020 09;7(1):559-587

Department of NanoEngineering, University of California, San Diego, La Jolla, California 92093, USA.

Viral nanotechnology exploits the prefabricated nanostructures of viruses, which are already abundant in nature. With well-defined molecular architectures, viral nanocarriers offer unprecedented opportunities for precise structural and functional manipulation using genetic engineering and/or bio-orthogonal chemistries. In this manner, they can be loaded with diverse molecular payloads for targeted delivery. Mammalian viruses are already established in the clinic for gene therapy and immunotherapy, and inactivated viruses or virus-like particles have long been used as vaccines. More recently, plant viruses and bacteriophages have been developed as nanocarriers for diagnostic imaging, vaccine and drug delivery, and combined diagnosis/therapy (theranostics). The first wave of these novel virus-based tools has completed clinical development and is poised to make an impact on clinical practice.
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http://dx.doi.org/10.1146/annurev-virology-010720-052252DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8018517PMC
September 2020

Doxorubicin-Loaded Physalis Mottle Virus Particles Function as a pH-Responsive Prodrug Enabling Cancer Therapy.

Biotechnol J 2020 Dec 8;15(12):e2000077. Epub 2020 Oct 8.

Department of NanoEngineering, University of California San Diego, 9500 Gilman Dr., La Jolla, CA, 92039, USA.

The controlled release of drugs using nanoparticle-based delivery vehicles is a promising strategy to improve the safety and efficacy of chemotherapy. A simple, scalable, and reproducible strategy is developed to synthesize a drug delivery system (DDS) by loading 6-maleimidocaproyl-hydrazone doxorubicin (DOX-EMCH) into the empty core of virus-like particles (VLPs) derived from Physalis mottle virus (PhMV) via a combination of chemical conjugation to cysteine residues and π-π stacking interactions with the anchored doxorubicin molecule. The DOX-EMCH prodrug features an acid-sensitive hydrazine linker that triggers the release of doxorubicin in the slightly acidic extracellular tumor microenvironment or acidic endosomal or lysosomal compartments following cellular uptake. The VLP external surface is coated with polyethylene glycol (PEG) to prevent non-specific uptake and improve biocompatibility. The DOX-PhMV-PEG particles are stable in vitro and show greater efficacy in vivo compared to free doxorubicin in a breast tumor mouse model (using MDA-MB-231 cells and nude mice): 92% of the tumor-bearing mice treated with DOX-PhMV-PEG are completely cured compared to 27% of those treated with free doxorubicin under the same conditions, representing a 3.4-fold improvement. These results lay a foundation for the further development of this biological drug delivery system for a new generation of chemotherapy products.
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http://dx.doi.org/10.1002/biot.202000077DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7888571PMC
December 2020

The unique potency of Cowpea mosaic virus (CPMV) cancer vaccine.

Biomater Sci 2020 Sep;8(19):5489-5503

Department of NanoEngineering, University of California-San Diego, La Jolla, CA 92039, USA. and Department of Bioengineering, University of California-San Diego, La Jolla, CA 92039, USA and Department of Radiology, University of California-San Diego, La Jolla, CA 92039, USA and Moores Cancer Center, University of California-San Diego, La Jolla, CA 92039, USA and Center for Nano-ImmunoEngineering, University of California-San Diego, La Jolla, CA 92039, USA.

The immunosuppressive tumor microenvironment enables cancer to resist immunotherapies. We have established that intratumoral administration of plant-derived Cowpea mosaic virus (CPMV) nanoparticles as an in situ vaccine overcomes the local immunosuppression and stimulates a potent anti-tumor response in several mouse cancer models and canine patients. CPMV does not infect mammalian cells but acts as a danger signal that leads to the recruitment and activation of innate and subsequently, adaptive immune cells. In the present study we addressed whether other icosahedral viruses or virus-like particles (VLPs) of plant, bacteriophage and mammalian origin can be similarly employed as intratumoral immunotherapy. Our results indicate that CPMV in situ vaccine outperforms Cowpea chlorotic mottle virus (CCMV), Physalis mosaic virus (PhMV), Sesbania mosaic virus (SeMV), bacteriophage Qβ VLPs, or Hepatitis B virus capsids (HBVc). Furthermore, ex vivo and in vitro assays reveal unique features of CPMV that makes it an inherently stronger immune stimulant.
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http://dx.doi.org/10.1039/d0bm01219jDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8086234PMC
September 2020

Intra- and intermolecular atomic-scale interactions in the receptor binding domain of SARS-CoV-2 spike protein: implication for ACE2 receptor binding.

Phys Chem Chem Phys 2020 Sep 5;22(33):18272-18283. Epub 2020 Aug 5.

Department of Physics and Astronomy, University of Missouri-Kansas City, Kansas City, Missouri, USA.

The COVID-19 pandemic poses a severe threat to human health with unprecedented social and economic disruption. Spike (S) glycoprotein in the SARS-CoV-2 virus is pivotal in understanding the virus anatomy, since it initiates the early contact with the ACE2 receptor in the human cell. The subunit S1 in chain A of S-protein has four structural domains: the receptor binding domain (RBD), the n-terminal domain (NTD) and two subdomains (SD1, SD2). We report details of the intra- and inter-molecular binding mechanism of RBD using density functional theory, including electronic structure, interatomic bonding and partial charge distribution. We identify five strong hydrogen bonds and analyze their roles in binding. This provides a pathway to a quantum-chemical understanding of the interaction between the S-protein and the ACE2 receptor with insights into the function of conserved features in the ACE2 receptor binding domain that could inform vaccine and drug development.
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http://dx.doi.org/10.1039/d0cp03145cDOI Listing
September 2020

COVID-19 vaccine development and a potential nanomaterial path forward.

Nat Nanotechnol 2020 08 15;15(8):646-655. Epub 2020 Jul 15.

Department of NanoEngineering, University of California San Diego, La Jolla, CA, USA.

The COVID-19 pandemic has infected millions of people with no clear signs of abatement owing to the high prevalence, long incubation period and lack of established treatments or vaccines. Vaccines are the most promising solution to mitigate new viral strains. The genome sequence and protein structure of the 2019-novel coronavirus (nCoV or SARS-CoV-2) were made available in record time, allowing the development of inactivated or attenuated viral vaccines along with subunit vaccines for prophylaxis and treatment. Nanotechnology benefits modern vaccine design since nanomaterials are ideal for antigen delivery, as adjuvants, and as mimics of viral structures. In fact, the first vaccine candidate launched into clinical trials is an mRNA vaccine delivered via lipid nanoparticles. To eradicate pandemics, present and future, a successful vaccine platform must enable rapid discovery, scalable manufacturing and global distribution. Here, we review current approaches to COVID-19 vaccine development and highlight the role of nanotechnology and advanced manufacturing.
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http://dx.doi.org/10.1038/s41565-020-0737-yDOI Listing
August 2020

Affinity of plant viral nanoparticle potato virus X (PVX) towards malignant B cells enables cancer drug delivery.

Biomater Sci 2020 Jul 17;8(14):3935-3943. Epub 2020 Jun 17.

Department of NanoEngineering, University of California San Diego, La Jolla, California 92093, USA.

Non-Hodgkin's B cell lymphomas (NHL) include a diverse set of neoplasms that constitute ∼90% of all lymphomas and the largest subset of blood cancers. While chemotherapy is the first line of treatment, the efficacy of contemporary chemotherapies is hampered by dose-limiting toxicities. Partly due to suboptimal dosing, ∼40% of patients exhibit relapsed or refractory disease. Therefore more efficacious drug delivery systems are urgently needed to improve survival of NHL patients. In this study we demonstrate a new drug delivery platform for NHL based on the plant virus Potato virus X (PVX). We observed a binding affinity of PVX towards malignant B cells. In a metastatic mouse model of NHL, we show that systemically administered PVX home to tissues harboring malignant B cells. When loaded with the chemotherapy monomethyl auristatin (MMAE), the PVX nanocarrier enables effective delivery of MMAE to human B lymphoma cells in a NHL mouse model leading to inhibition of lymphoma growth in vivo and improved survival. Thus, PVX nanoparticle is a promising drug delivery platform for B cell malignancies.
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http://dx.doi.org/10.1039/d0bm00683aDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7810362PMC
July 2020

Viral nanoparticles for drug delivery, imaging, immunotherapy, and theranostic applications.

Adv Drug Deliv Rev 2020 27;156:214-235. Epub 2020 Jun 27.

Department of Bioengineering, University of California-San Diego, La Jolla, CA 92093, United States; Department of NanoEngineering, University of California-San Diego, La Jolla, CA 92093, United States; Department of Radiology, University of California-San Diego, La Jolla, CA 92093, United States; Moores Cancer Center, University of California-San Diego, La Jolla, CA 92093, United States; Center for Nano-ImmunoEngineering, University of California-San Diego, La Jolla, CA 92093, United States. Electronic address:

Viral nanoparticles (VNPs) encompass a diverse array of naturally occurring nanomaterials derived from plant viruses, bacteriophages, and mammalian viruses. The application and development of VNPs and their genome-free versions, the virus-like particles (VLPs), for nanomedicine is a rapidly growing. VLPs can encapsulate a wide range of active ingredients as well as be genetically or chemically conjugated to targeting ligands to achieve tissue specificity. VLPs are manufactured through scalable fermentation or molecular farming, and the materials are biocompatible and biodegradable. These properties have led to a wide range of applications, including cancer therapies, immunotherapies, vaccines, antimicrobial therapies, cardiovascular therapies, gene therapies, as well as imaging and theranostics. The use of VLPs as drug delivery agents is evolving, and sufficient research must continuously be undertaken to translate these therapies to the clinic. This review highlights some of the novel research efforts currently underway in the VNP drug delivery field in achieving this greater goal.
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http://dx.doi.org/10.1016/j.addr.2020.06.024DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7320870PMC
June 2020

Protein cages and virus-like particles: from fundamental insight to biomimetic therapeutics.

Biomater Sci 2020 May;8(10):2771-2777

Centre for Cell Factories and Biopolymers, Griffith Institute for Drug Discovery, Griffith University, Nathan, QLD 4111, Australia. and Synthetic Biology Future Science Platform, Commonwealth Scientific and Industrial Research Organisation (CSIRO), Brisbane, QLD 4001, Australia.

Protein cages (viral and non-viral) found in nature have evolved for a variety of purposes and are found in all kingdoms of life. The main functions of these nanoscale compartments are the protection and delivery of nucleic acids e.g. virus capsids, or the enrichment and sequestration of metabolons e.g. bacterial microcompartments. This review focuses on recent developments of protein cages for use in immunotherapy and therapeutic delivery. In doing so, we highlight the unique ways in which protein cages have informed on fundamental principles governing bio-nano interactions. With the enormous existing design space among naturally occurring protein cages, there is still much to learn from studying them as biomimetic particles.
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http://dx.doi.org/10.1039/d0bm00159gDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8085892PMC
May 2020

Active Delivery of VLPs Promotes Anti-Tumor Activity in a Mouse Ovarian Tumor Model.

Small 2020 05 24;16(20):e1907150. Epub 2020 Apr 24.

Department of NanoEngineering, University of California San Diego, La Jolla, CA, 92093, USA.

Virus-like nanoparticles (VLPs) have been used as an attractive means in cancer immunotherapy because of their unique intrinsic immunostimulatory properties. However, for treating metastatic tumors in the peritoneal cavity, such as ovarian cancer, multiple injections of therapy are needed due to the large peritoneal space and fast excretion of therapy. Here, it is reported on the development of active VLP delivery vehicles for the treatment of peritoneal ovarian tumors using biocompatible Qβ VLPs-loaded Mg-based micromotors. The autonomous propulsion of such Qβ VLPs-loaded Mg-micromotors in the peritoneal fluid enables active delivery of intact immunostimulatory Qβ VLPs to the peritoneal space of ovarian tumor bearing mice, greatly enhancing the local distribution and retention of Qβ VLPs. Such improved distribution and longer retention time of Qβ in the peritoneal cavity leads to enhanced immunostimulation and therefore increased survival rate of tumor-bearing mice compared to a passive Qβ treatment. For clinical translation, the active delivery of VLPs holds great promise for tumor immunotherapy toward the treatment of different types of primary and metastatic tumors in the peritoneal cavity.
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http://dx.doi.org/10.1002/smll.201907150DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7507763PMC
May 2020

Site-Specific Antibody Conjugation Strategy to Functionalize Virus-Based Nanoparticles.

Bioconjug Chem 2020 05 13;31(5):1408-1416. Epub 2020 Apr 13.

Amine/thiol-reactive chemistries are commonly used to conjugate antibodies to pharmaceuticals or nanoparticles. Yet, these conjugation strategies often result in unfavorable outcomes such as heterogeneous antibody display with hindered biological activity or aggregation due to multivalent interactions of the antibody and nanoparticles. Here, we report the application of a site-specific and enzymatically driven antibody conjugation strategy to functionalize virus-based nanoparticles (VNPs). Specifically, an azide-handle was introduced into the Fc region of a set of immunoglobulins using a two-step enzymatic reaction: (1) cleavage of -linked glycan in the Fc region by a glycosidase and (2) conjugation of a chemically reactive linker (containing an azide functional handle) using a microbial transglutaminase. Conjugation of the azide-functional antibodies to several VNPs was achieved by making use of strain-promoted azide-alkyne cycloaddition. We report the conjugation of three immunoglobulin (IgG) isotypes (human IgG from sera, anti-CD47 Rat IgG2a, κ, and Trastuzumab recombinant humanized IgG1, κ) to the plant virus cowpea mosaic virus (CPMV) and the lysine mutant of tobacco mosaic virus (TMVlys) as well as bacteriophage Qβ. Site-specific conjugation resulted in stable and functional antibody-VNP conjugates. In stark contrast, the use of heterobifunctional linkers targeting thiols and amines on the antibodies and VNPs, respectively, led to aggregation due to nonspecific and multivalent coupling between the antibodies and VNPs. We demonstrate that antibody-VNP conjugates were functional, and Trastuzumab-displaying VNPs targeted HER2-positive SKOV-3 human ovarian cancer cells. This bioconjugation strategy adds to the portfolio of methods that can be used for designing functional antibody-VNP conjugates.
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http://dx.doi.org/10.1021/acs.bioconjchem.0c00118DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8085887PMC
May 2020

S100A9-targeted tobacco mosaic virus nanoparticles exhibit high specificity toward atherosclerotic lesions in ApoE mice.

J Mater Chem B 2019 03 13;7(11):1842-1846. Epub 2019 Feb 13.

Department of Nanoengineering, University of California San Diego, La Jolla, CA 92093, USA.

We integrate a biocompatible plant virus-based nanotechnology (tobacco mosaic virus, TMV) with S100A9-targeting peptides for its application in imaging and diagnosis of atherosclerosis. S100A9-targeted TMV nanoparticles exhibit remarkable specificity to S100A9 and targeting of atherosclerosis lesions in ApoE mice.
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http://dx.doi.org/10.1039/c8tb02276cDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7147689PMC
March 2019

Antibody Response against Cowpea Mosaic Viral Nanoparticles Improves Vaccine Efficacy in Ovarian Cancer.

ACS Nano 2020 03 5;14(3):2994-3003. Epub 2020 Mar 5.

Cancer immunotherapies are designed to facilitate recognition and elimination of transformed cells by the immune system. We have established the immunotherapeutic efficacy of the plant virus cowpea mosaic virus (CPMV) as an vaccine in several syngeneic tumor mouse models as well as in companion dogs with metastatic melanoma. Intratumoral injection of CPMV modulates the local tumor microenvironment to relieve immunosuppression and potentiate antitumor immunity. The viral nucleocapsid that drives this antitumor immunity, however, also is a potent immunogen itself, and thus immune response in the form of anti-CPMV antibodies is expected during the treatment based on repeat administrations. Moreover, being part of the food chain, pre-existing antibodies to plant viruses may be prevalent. The presence of such pre-existing anti-CPMV immunity could potentially impact immunotherapeutic efficacy of the vaccine and could have translational implications. To address such concerns, this study evaluated the efficacy of CPMV vaccine in the presence of pre-existing antibodies in a syngeneic mouse model of ovarian cancer. Our results indicate that prior exposure to CPMV had no negative impact on the efficacy of CPMV vaccine. Strikingly, an improved efficacy of CPMV vaccine was observed. This study therefore presents an important milestone in the translational development of plant viral-based vaccines and alleviates concerns about the presence of anti-CPMV antibodies, which are developed during the course of treatment but have no impact on immunotherapeutic efficacy.
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http://dx.doi.org/10.1021/acsnano.9b07865DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8085886PMC
March 2020

Nanocarriers for the Delivery of Medical, Veterinary, and Agricultural Active Ingredients.

ACS Nano 2020 03 3;14(3):2678-2701. Epub 2020 Mar 3.

Nanocarrier-based delivery systems can be used to increase the safety and efficacy of active ingredients in medical, veterinary, or agricultural applications, particularly when such ingredients are unstable, sparingly soluble, or cause off-target effects. In this review, we highlight the diversity of nanocarrier materials and their key advantages compared to free active ingredients. We discuss current trends based on peer-reviewed research articles, patent applications, clinical trials, and the nanocarrier formulations already approved by regulatory bodies. Although most nanocarriers have been engineered to combat cancer, the number of formulations developed for other purposes is growing rapidly, especially those for the treatment of infectious diseases and parasites affecting humans, livestock, and companion animals. The regulation and prohibition of many pesticides have also fueled research to develop targeted pesticide delivery systems based on nanocarriers, which maximize efficacy while minimizing the environmental impact of agrochemicals.
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http://dx.doi.org/10.1021/acsnano.0c00173DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8085836PMC
March 2020

Dynamic, Simultaneous Concentration Mapping of Multiple MRI Contrast Agents with Dual Contrast - Magnetic Resonance Fingerprinting.

Sci Rep 2019 12 27;9(1):19888. Epub 2019 Dec 27.

Department of Radiology, Case Western Reserve University, Cleveland, OH, USA.

Synchronous assessment of multiple MRI contrast agents in a single scanning session would provide a new "multi-color" imaging capability similar to fluorescence imaging but with high spatiotemporal resolution and unlimited imaging depth. This multi-agent MRI technology would enable a whole new class of basic science and clinical MRI experiments that simultaneously explore multiple physiologic/molecular events in vivo. Unfortunately, conventional MRI acquisition techniques are only capable of detecting and quantifying one paramagnetic MRI contrast agent at a time. Herein, the Dual Contrast - Magnetic Resonance Fingerprinting (DC-MRF) methodology was extended for in vivo application and evaluated by simultaneously and dynamically mapping the intra-tumoral concentration of two MRI contrast agents (Gd-BOPTA and Dy-DOTA-azide) in a mouse glioma model. Co-registered gadolinium and dysprosium concentration maps were generated with sub-millimeter spatial resolution and acquired dynamically with just over 2-minute temporal resolution. Mean tumor Gd and Dy concentration measurements from both single agent and dual agent DC-MRF studies demonstrated significant correlations with ex vivo mass spectrometry elemental analyses. This initial in vivo study demonstrates the potential for DC-MRF to provide a useful dual-agent MRI platform.
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http://dx.doi.org/10.1038/s41598-019-56531-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6934650PMC
December 2019

Built-In Active Microneedle Patch with Enhanced Autonomous Drug Delivery.

Adv Mater 2020 Jan 4;32(1):e1905740. Epub 2019 Nov 4.

Department of Nanoengineering, University of California San Diego, La Jolla, CA, 92093, USA.

The use of microneedles has facilitated the painless localized delivery of drugs across the skin. However, their efficacy has been limited by slow diffusion of molecules and often requires external triggers. Herein, an autonomous and degradable, active microneedle delivery platform is introduced, employing magnesium microparticles loaded within the microneedle patch, as the built-in engine for deeper and faster intradermal payload delivery. The magnesium particles react with the interstitial fluid, leading to an explosive-like rapid production of H bubbles, providing the necessary force to breach dermal barriers and enhance payload delivery. The release kinetics of active microneedles is evaluated in vitro by measuring the amount of IgG antibody (as a model drug) that passed through phantom tissue and a pigskin barrier. In vivo experiments using a B16F10 mouse melanoma model demonstrate that the active delivery of anti-CTLA-4 (a checkpoint inhibitor drug) results in greatly enhanced immune response and significantly longer survival. Moreover, spatially resolved zones of active and passive microneedles allow a combinatorial rapid burst response along with slow, sustained release, respectively. Such versatile and effective autonomous dynamic microneedle delivery technology offers considerable promise for a wide range of therapeutic applications, toward a greatly enhanced outcome, convenience, and cost.
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http://dx.doi.org/10.1002/adma.201905740DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7014935PMC
January 2020

Endosomal toll-like receptors play a key role in activation of primary human monocytes by cowpea mosaic virus.

Immunology 2020 02 15;159(2):183-192. Epub 2019 Nov 15.

Division of Infectious Diseases and HIV Medicine, School of Medicine, Case Western Reserve University, Cleveland, OH, USA.

The plant virus, cowpea mosaic virus (CPMV), has demonstrated a remarkable capacity to induce anti-tumour immune responses following direct administration into solid tumours. The molecular pathways that account for these effects and the capacity of CPMV to activate human cells are not well defined. Here, we examine the ability of CPMV particles to activate human monocytes, dendritic cells (DCs) and macrophages. Monocytes in peripheral blood mononuclear cell cultures and purified CD14+ monocytes were readily activated by CPMV in vitro, leading to induction of HLA-DR, CD86, PD-L1, IL-15R and CXCL10 expression. Monocytes released chemokines, CXCL10, MIP-1α and MIP-1β into cell culture supernatants after incubation with CPMV. DC subsets (pDC and mDC) and monocyte-derived macrophages also demonstrated evidence of activation after incubation with CPMV. Inhibitors of spleen tyrosine kinase (SYK), endocytosis or endocytic acidification impaired the capacity of CPMV to activate monocytes. Furthermore, CPMV activation of monocytes was partially blocked by a TLR7/8 antagonist. These data demonstrate that CPMV activates human monocytes in a manner dependent on SYK signalling, endosomal acidification and with an important contribution from TLR7/8 recognition.
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http://dx.doi.org/10.1111/imm.13135DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6954739PMC
February 2020