scholarly journals Design of Alphavirus Virus-Like Particles Presenting Circumsporozoite Junctional Epitopes That Elicit Protection against Malaria

Vaccines ◽  
2021 ◽  
Vol 9 (3) ◽  
pp. 272
Author(s):  
Joseph R. Francica ◽  
Wei Shi ◽  
Gwo-Yu Chuang ◽  
Steven J. Chen ◽  
Lais Da Silva Pereira ◽  
...  
Keyword(s):  
Malaria Vaccine ◽  
Structural Integrity ◽  
Keyhole Limpet Hemocyanin ◽  
Effective Vaccine ◽  
Virus Like Particles ◽  
Human Antibodies ◽  
Negative Stain ◽  
Serum Titer ◽  
Negative Stain Electron Microscopy ◽  
Genetic Fusion

The most advanced malaria vaccine, RTS,S, includes the central repeat and C-terminal domains of the Plasmodium falciparum circumsporozoite protein (PfCSP). We have recently isolated human antibodies that target the junctional region between the N-terminal and repeat domains that are not included in RTS,S. Due to the fact that these antibodies protect against malaria challenge in mice, their epitopes could be effective vaccine targets. Here, we developed immunogens displaying PfCSP junctional epitopes by genetic fusion to either the N-terminus or B domain loop of the E2 protein from chikungunya (CHIK) alphavirus and produced CHIK virus-like particles (CHIK-VLPs). The structural integrity of these junctional-epitope–CHIK-VLP immunogens was confirmed by negative-stain electron microscopy. Immunization of these CHIK-VLP immunogens reduced parasite liver load by up to 95% in a mouse model of malaria infection and elicited better protection than when displayed on keyhole limpet hemocyanin, a commonly used immunogenic carrier. Protection correlated with PfCSP serum titer. Of note, different junctional sequences elicited qualitatively different reactivities to overlapping PfCSP peptides. Overall, these results show that the junctional epitopes of PfCSP can induce protective responses when displayed on CHIK-VLP immunogens and provide a basis for the development of a next generation malaria vaccine to expand the breadth of anti-PfCSP immunity.

scholarly journals Quality Assurance Testing of Papilloma Virus-Like Particles by Negative Stain Electron Microscopy

2011 ◽  
Vol 17 (S2) ◽  
pp. 288-289
Author(s):  
C Humphrey ◽  
M Metcalfe ◽  
I Rajbhandari ◽  
C Kryston ◽  
G Panicker ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Quality Assurance ◽  
Papilloma Virus ◽  
Virus Like Particles ◽  
Negative Stain ◽  
Negative Stain Electron Microscopy

Extended abstract of a paper presented at Microscopy and Microanalysis 2011 in Nashville, Tennessee, USA, August 7–August 11, 2011.

scholarly journals Newcastle Disease Virus-Like Particles Displaying Prefusion-Stabilized SARS-CoV-2 Spikes Elicit Potent Neutralizing Responses

Vaccines ◽  
2021 ◽  
Vol 9 (2) ◽  
pp. 73 ◽  
Author(s):  
Yongping Yang ◽  
Wei Shi ◽  
Olubukola M. Abiona ◽  
Alexandra Nazzari ◽  
Adam S. Olia ◽  
...  
Keyword(s):  
Newcastle Disease Virus ◽  
Disease Virus ◽  
Newcastle Disease ◽  
Neutralizing Antibodies ◽  
Geometric Mean ◽  
Virus Like Particles ◽  
Negative Stain ◽  
Molar Equivalent ◽  
Boost Immunization ◽  
Negative Stain Electron Microscopy

The COVID-19 pandemic highlights an urgent need for vaccines that confer protection from SARS-CoV-2 infection. One approach to an effective COVID-19 vaccine may be through the display of SARS-CoV-2 spikes on the surface of virus-like particles, in a manner structurally mimicking spikes on a native virus. Here we report the development of Newcastle disease virus-like particles (NDVLPs) displaying the prefusion-stabilized SARS-CoV-2 spike ectodomain (S2P). Immunoassays with SARS-CoV-2-neutralizing antibodies revealed the antigenicity of S2P-NDVLP to be generally similar to that of soluble S2P, and negative-stain electron microscopy showed S2P on the NDVLP surface to be displayed with a morphology corresponding to its prefusion conformation. Mice immunized with S2P-NDVLP showed substantial neutralization titers (geometric mean ID50 = 386) two weeks after prime immunization, significantly higher than those elicited by a molar equivalent amount of soluble S2P (geometric mean ID50 = 17). Neutralizing titers at Week 5, two weeks after a boost immunization with S2P-NDVLP doses ranging from 2.0 to 250 μg, extended from 2125 to 4552, and these generally showed a higher ratio of neutralization versus ELISA than observed with soluble S2P. Overall, S2P-NDVLP appears to be a promising COVID-19 vaccine candidate capable of eliciting substantial neutralizing activity.

scholarly journals Second messenger Ap4A polymerizes target protein HINT1 to transduce signals in FcεRI-activated mast cells

2019 ◽  
Vol 10 (1) ◽  
Author(s):  
Jing Yu ◽  
Zaizhou Liu ◽  
Yuanyuan Liang ◽  
Feng Luo ◽  
Jie Zhang ◽  
...  
Keyword(s):  
Mast Cells ◽  
Transcriptional Activation ◽  
Second Messenger ◽  
Signaling Mechanism ◽  
Cellular Processes ◽  
Negative Stain ◽  
Competitive Mechanism ◽  
Rat Basophilic Leukemia Cells ◽  
Negative Stain Electron Microscopy ◽  
Basophilic Leukemia

Abstract Signal transduction systems enable organisms to monitor their external environments and accordingly adjust the cellular processes. In mast cells, the second messenger Ap4A binds to the histidine triad nucleotide-binding protein 1 (HINT1), disrupts its interaction with the microphthalmia-associated transcription factor (MITF), and eventually activates the transcription of genes downstream of MITF in response to immunostimulation. How the HINT1 protein recognizes and is regulated by Ap4A remain unclear. Here, using eight crystal structures, biochemical experiments, negative stain electron microscopy, and cellular experiments, we report that Ap4A specifically polymerizes HINT1 in solution and in activated rat basophilic leukemia cells. The polymerization interface overlaps with the area on HINT1 for MITF interaction, suggesting a possible competitive mechanism to release MITF for transcriptional activation. The mechanism depends precisely on the length of the phosphodiester linkage of Ap4A. These results highlight a direct polymerization signaling mechanism by the second messenger.

scholarly journals Structure–function mapping of a heptameric module in the nuclear pore complex

2012 ◽  
Vol 196 (4) ◽  
pp. 419-434 ◽  
Author(s):  
Javier Fernandez-Martinez ◽  
Jeremy Phillips ◽  
Matthew D. Sekedat ◽  
Ruben Diaz-Avalos ◽  
Javier Velazquez-Muriel ◽  
...  
Keyword(s):  
Nuclear Pore Complex ◽  
Protein Domain ◽  
Integrative Approach ◽  
Nuclear Pore ◽  
Mapping Data ◽  
Phenotypic Data ◽  
Negative Stain ◽  
Domain Mapping ◽  
Gene Duplication And Loss ◽  
Negative Stain Electron Microscopy

The nuclear pore complex (NPC) is a multiprotein assembly that serves as the sole mediator of nucleocytoplasmic exchange in eukaryotic cells. In this paper, we use an integrative approach to determine the structure of an essential component of the yeast NPC, the ∼600-kD heptameric Nup84 complex, to a precision of ∼1.5 nm. The configuration of the subunit structures was determined by satisfaction of spatial restraints derived from a diverse set of negative-stain electron microscopy and protein domain–mapping data. Phenotypic data were mapped onto the complex, allowing us to identify regions that stabilize the NPC’s interaction with the nuclear envelope membrane and connect the complex to the rest of the NPC. Our data allow us to suggest how the Nup84 complex is assembled into the NPC and propose a scenario for the evolution of the Nup84 complex through a series of gene duplication and loss events. This work demonstrates that integrative approaches based on low-resolution data of sufficient quality can generate functionally informative structures at intermediate resolution.

scholarly journals Negative Stain Transmission Electron Microscopy of Viruses and Virus-like Particles

2009 ◽  
Vol 15 (S2) ◽  
pp. 376-377 ◽  
Author(s):  
C Humphrey
Keyword(s):  
Electron Microscopy ◽  
Transmission Electron Microscopy ◽  
Virus Like Particles ◽  
Negative Stain ◽  
Transmission Electron

Extended abstract of a paper presented at Microscopy and Microanalysis 2009 in Richmond, Virginia, USA, July 26 – July 30, 2009

scholarly journals Temperature-dependent reversible assembly of taxol-treated microtubules.

1987 ◽  
Vol 105 (6) ◽  
pp. 2847-2854 ◽  
Author(s):  
C A Collins ◽  
R B Vallee
Keyword(s):  
Electron Microscopy ◽  
Self Assembly ◽  
Temperature Dependent ◽  
Reverse Phase Hplc ◽  
Reverse Phase ◽  
Microtubule Associated Proteins ◽  
Negative Stain ◽  
Microtubule Protein ◽  
Associated Proteins ◽  
Negative Stain Electron Microscopy

Taxol is a plant alkaloid that binds to and strongly stabilizes microtubules. Taxol-treated microtubules resist depolymerization under a variety of conditions that readily disassemble untreated microtubules. We report here that taxol-treated microtubules can be induced to disassemble by a combination of depolymerizating conditions. Reversible cycles of disassembly and reassembly were carried out using taxol-containing microtubules from calf brain and sea urchin eggs by shifting temperature in the presence of millimolar levels of Ca2+. Microtubules depolymerized completely, yielding dimers and ring-shaped oligomers as revealed by negative stain electron microscopy and Bio-Gel A-15m chromatography, and reassembled into well-formed microtubule polymer structures. Microtubule-associated proteins (MAPs), including species previously identified only by taxol-based purification such as MAP 1B and kinesin, were found to copurify with tubulin through reversible assembly cycles. To determine whether taxol remained bound to tubulin subunits, we subjected depolymerized taxol-treated microtubule protein to Sephadex G-25 chromatography, and the fractions were assayed for taxol content by reverse-phase HPLC. Taxol was found to be dissociated from the depolymerized microtubules. Protein treated in this way was found to be competent to reassemble, but now required conditions comparable with those for protein that had never been exposed to taxol. Thus, the binding of taxol to tubulin can be reversed. This has implications for the mechanism of taxol action and for the purification of microtubules from a wide variety of sources for use in self-assembly experiments.

scholarly journals A site of varicella-zoster virus vulnerability identified by structural studies of neutralizing antibodies bound to the glycoprotein complex gHgL

2015 ◽  
Vol 112 (19) ◽  
pp. 6056-6061 ◽  
Author(s):  
Yi Xing ◽  
Stefan L. Oliver ◽  
TuongVi Nguyen ◽  
Claudio Ciferri ◽  
Avishek Nandi ◽  
...  
Keyword(s):  
Varicella Zoster Virus ◽  
Neutralizing Antibodies ◽  
Mutational Analysis ◽  
Natural Immunity ◽  
Varicella Zoster ◽  
Glycoprotein Complex ◽  
Negative Stain ◽  
Virion Envelope ◽  
Negative Stain Electron Microscopy ◽  
A Site

Varicella-zoster virus (VZV), of the familyAlphaherpesvirinae, causes varicella in children and young adults, potentially leading to herpes zoster later in life on reactivation from latency. The conserved herpesvirus glycoprotein gB and the heterodimer gHgL mediate virion envelope fusion with cell membranes during virus entry. Naturally occurring neutralizing antibodies against herpesviruses target these entry proteins. To determine the molecular basis for VZV neutralization, crystal structures of gHgL were determined in complex with fragments of antigen binding (Fabs) from two human monoclonal antibodies, IgG-94 and IgG-RC, isolated from seropositive subjects. These structures reveal that the antibodies target the same site, composed of residues from both gH and gL, distinct from two other neutralizing epitopes identified by negative-stain electron microscopy and mutational analysis. Inhibition of gB/gHgL-mediated membrane fusion and structural comparisons with herpesvirus homologs suggest that the IgG-RC/94 epitope is in proximity to the site on VZV gHgL that activates gB. Immunization studies proved that the anti-gHgL IgG-RC/94 epitope is a critical target for antibodies that neutralize VZV. Thus, the gHgL/Fab structures delineate a site of herpesvirus vulnerability targeted by natural immunity.

scholarly journals Phase I Testing of a Malaria Vaccine Composed of Hepatitis B Virus Core Particles Expressing Plasmodium falciparum Circumsporozoite Epitopes

2004 ◽  
Vol 72 (11) ◽  
pp. 6519-6527 ◽  
Author(s):  
Elizabeth H. Nardin ◽  
Giane A. Oliveira ◽  
J. Mauricio Calvo-Calle ◽  
Kristiane Wetzel ◽  
Carolin Maier ◽  
...  
Keyword(s):  
Plasmodium Falciparum ◽  
Hepatitis B Virus ◽  
Hepatitis B ◽  
Phase I ◽  
Malaria Vaccine ◽  
Vaccine Candidate ◽  
Virus Like Particles ◽  
Virus Core ◽  
B Virus ◽  
Malaria Vaccine Candidate

ABSTRACT We report the first phase I trial to assess the safety and immunogenicity of a malaria vaccine candidate, ICC-1132 (Malarivax), composed of a modified hepatitis B virus core protein (HBc) containing minimal epitopes of the Plasmodium falciparum circumsporozoite (CS) protein. When expressed in Escherichia coli, the recombinant ICC-1132 protein forms virus-like particles that were found to be highly immunogenic in preclinical studies of mice and monkeys. Twenty healthy adult volunteers received a 20- or a 50-μg dose of alum-adsorbed ICC-1132 administered intramuscularly at 0, 2, and 6 months. The majority of volunteers in the group receiving the 50-μg dose developed antibodies to CS repeats as well as to HBc. Malaria-specific T cells that secreted gamma interferon were also detected after a single immunization with ICC-1132-alum. These studies support ICC-1132 as a promising malaria vaccine candidate for further clinical testing using more-potent adjuvant formulations and confirm the potential of modified HBc virus-like particles as a delivery platform for vaccines against other human pathogens.

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