Metabolic engineering of HEK293 cells to improve transient transfection and cell budding of HIV‐1 virus‐like particles

2021 ◽  
Author(s):  
Jesús Lavado‐García ◽  
Andy Díaz‐Maneh ◽  
Núria Canal‐Paulí ◽  
Pol Pérez‐Rubio ◽  
Francesc Gòdia ◽  
...  
Keyword(s):  
Metabolic Engineering ◽  
Transient Transfection ◽  
Hek293 Cells ◽  
Virus Like Particles ◽  
Hiv 1

Optimized production of HIV-1 virus-like particles by transient transfection in CAP-T cells

2015 ◽  
Vol 100 (9) ◽  
pp. 3935-3947 ◽  
Author(s):  
Sonia Gutiérrez-Granados ◽  
Laura Cervera ◽  
María de las Mercedes Segura ◽  
Jens Wölfel ◽  
Francesc Gòdia
Keyword(s):  
T Cells ◽  
Transient Transfection ◽  
Virus Like Particles ◽  
Hiv 1

scholarly journals Immunogenicity of HIV-1-Based Virus-Like Particles with Increased Incorporation and Stability of Membrane-Bound Env

Vaccines ◽  
2021 ◽  
Vol 9 (3) ◽  
pp. 239
Author(s):  
Christopher A. Gonelli ◽  
Hannah A. D. King ◽  
Charlene Mackenzie ◽  
Secondo Sonza ◽  
Rob J. Center ◽  
...  
Keyword(s):  
Neutralizing Antibody ◽  
Antibody Responses ◽  
Neutralization Activity ◽  
Virus Like Particles ◽  
Antibody Isotypes ◽  
Immunodeficiency Virus ◽  
Proline Substitution ◽  
Membrane Bound ◽  
Antibody Epitopes ◽  
Hiv 1

An optimal prophylactic vaccine to prevent human immunodeficiency virus (HIV-1) transmission should elicit protective antibody responses against the HIV-1 envelope glycoprotein (Env). Replication-incompetent HIV-1 virus-like particles (VLPs) offer the opportunity to present virion-associated Env with a native-like structure during vaccination that closely resembles that encountered on infectious virus. Here, we optimized the incorporation of Env into previously designed mature-form VLPs (mVLPs) and assessed their immunogenicity in mice. The incorporation of Env into mVLPs was increased by replacing the Env transmembrane and cytoplasmic tail domains with those of influenza haemagglutinin (HA-TMCT). Furthermore, Env was stabilized on the VLP surface by introducing an interchain disulfide and proline substitution (SOSIP) mutations typically employed to stabilize soluble Env trimers. The resulting mVLPs efficiently presented neutralizing antibody epitopes while minimizing exposure of non-neutralizing antibody sites. Vaccination of mice with mVLPs elicited a broader range of Env-specific antibody isotypes than Env presented on immature VLPs or extracellular vesicles. The mVLPs bearing HA-TMCT-modified Env consistently induced anti-Env antibody responses that mediated modest neutralization activity. These mVLPs are potentially useful immunogens for eliciting neutralizing antibody responses that target native Env epitopes on infectious HIV-1 virions.

Characterization of HIV‐1 virus‐like particles and determination of Gag stoichiometry for different production platforms

2021 ◽  
Author(s):  
Jesús Lavado‐García ◽  
Inmaculada Jorge ◽  
Arnau Boix‐Besora ◽  
Jesús Vázquez ◽  
Francesc Gòdia ◽  
...  
Keyword(s):  
Virus Like Particles ◽  
Hiv 1

scholarly journals Effect of Small Polyanions on In Vitro Assembly of Selected Members of Alpha-, Beta- and Gammaretroviruses

Viruses ◽  
2021 ◽  
Vol 13 (1) ◽  
pp. 129
Author(s):  
Alžběta Dostálková ◽  
Barbora Vokatá ◽  
Filip Kaufman ◽  
Pavel Ulbrich ◽  
Tomáš Ruml ◽  
...  
Keyword(s):  
Equine Infectious Anemia Virus ◽  
Electron Tomography ◽  
Leukemia Virus ◽  
Virus Like Particles ◽  
Rous Sarcoma ◽  
Sarcoma Virus ◽  
Anemia Virus ◽  
Immature Virus ◽  
Hiv 1

The assembly of a hexameric lattice of retroviral immature particles requires the involvement of cell factors such as proteins and small molecules. A small, negatively charged polyanionic molecule, myo-inositol hexaphosphate (IP6), was identified to stimulate the assembly of immature particles of HIV-1 and other lentiviruses. Interestingly, cryo-electron tomography analysis of the immature particles of two lentiviruses, HIV-1 and equine infectious anemia virus (EIAV), revealed that the IP6 binding site is similar. Based on this amino acid conservation of the IP6 interacting site, it is presumed that the assembly of immature particles of all lentiviruses is stimulated by IP6. Although this specific region for IP6 binding may be unique for lentiviruses, it is plausible that other retroviral species also recruit some small polyanion to facilitate the assembly of their immature particles. To study whether the assembly of retroviruses other than lentiviruses can be stimulated by polyanionic molecules, we measured the effect of various polyanions on the assembly of immature virus-like particles of Rous sarcoma virus (RSV), a member of alpharetroviruses, Mason-Pfizer monkey virus (M-PMV) representative of betaretroviruses, and murine leukemia virus (MLV), a member of gammaretroviruses. RSV, M-PMV and MLV immature virus-like particles were assembled in vitro from truncated Gag molecules and the effect of selected polyanions, myo-inostol hexaphosphate, myo-inositol, glucose-1,6-bisphosphate, myo-inositol hexasulphate, and mellitic acid, on the particles assembly was quantified. Our results suggest that the assembly of immature particles of RSV and MLV was indeed stimulated by the presence of myo-inostol hexaphosphate and myo-inositol, respectively. In contrast, no effect on the assembly of M-PMV as a betaretrovirus member was observed.

scholarly journals A Conformational Transition Observed in Single HIV-1 Gag Molecules during In Vitro Assembly of Virus-Like Particles

2014 ◽  
Vol 88 (6) ◽  
pp. 3577-3585 ◽  
Author(s):  
J. B. Munro ◽  
A. Nath ◽  
M. Farber ◽  
S. A. K. Datta ◽  
A. Rein ◽  
...  
Keyword(s):  
Conformational Transition ◽  
Virus Like Particles ◽  
In Vitro Assembly ◽  
Hiv 1

scholarly journals Helical Conformation in the CA-SP1 Junction of the Immature HIV-1 Lattice Determined from Solid-State NMR of Virus-like Particles

2016 ◽  
Vol 138 (37) ◽  
pp. 12029-12032 ◽  
Author(s):  
Marvin J. Bayro ◽  
Barbie K. Ganser-Pornillos ◽  
Kaneil K. Zadrozny ◽  
Mark Yeager ◽  
Robert Tycko
Keyword(s):  
Solid State ◽  
Solid State Nmr ◽  
Helical Conformation ◽  
Virus Like Particles ◽  
Hiv 1

Long-term follow-up: no effect of therapeutic vaccination with HIV-1 p17/p24:Ty virus-like particles on HIV-1 disease progression

Vaccine ◽  
2002 ◽  
Vol 20 (17-18) ◽  
pp. 2343-2347 ◽  
Author(s):  
Catharina E.A. Lindenburg ◽  
Ineke Stolte ◽  
Miranda W. Langendam ◽  
Frank Miedema ◽  
Ian G. Williams ◽  
...  
Keyword(s):  
Disease Progression ◽  
Therapeutic Vaccination ◽  
Virus Like Particles ◽  
Long Term Follow Up ◽  
Hiv 1

scholarly journals The Retroviral Capsid Domain Dictates Virion Size, Morphology, and Coassembly of Gag into Virus-Like Particles

2005 ◽  
Vol 79 (21) ◽  
pp. 13463-13472 ◽  
Author(s):  
Danso Ako-Adjei ◽  
Marc C. Johnson ◽  
Volker M. Vogt
Keyword(s):  
Nuclear Export ◽  
Leukemia Virus ◽  
Particle Diameter ◽  
Particle Morphology ◽  
Structural Protein ◽  
Wild Type ◽  
Virus Like Particles ◽  
Gag Proteins ◽  
Hiv 1

ABSTRACT The retroviral structural protein, Gag, is capable of independently assembling into virus-like particles (VLPs) in living cells and in vitro. Immature VLPs of human immunodeficiency virus type 1 (HIV-1) and of Rous sarcoma virus (RSV) are morphologically distinct when viewed by transmission electron microscopy (TEM). To better understand the nature of the Gag-Gag interactions leading to these distinctions, we constructed vectors encoding several RSV/HIV-1 chimeric Gag proteins for expression in either insect cells or vertebrate cells. We used TEM, confocal fluorescence microscopy, and a novel correlative scanning EM (SEM)-confocal microscopy technique to study the assembly properties of these proteins. Most chimeric proteins assembled into regular VLPs, with the capsid (CA) domain being the primary determinant of overall particle diameter and morphology. The presence of domains between matrix and CA also influenced particle morphology by increasing the spacing between the inner electron-dense ring and the VLP membrane. Fluorescently tagged versions of wild-type RSV, HIV-1, or murine leukemia virus Gag did not colocalize in cells. However, wild-type Gag proteins colocalized extensively with chimeric Gag proteins bearing the same CA domain, implying that Gag interactions are mediated by CA. A dramatic example of this phenomenon was provided by a nuclear export-deficient chimera of RSV Gag carrying the HIV-1 CA domain, which by itself localized to the nucleus but relocalized to the cytoplasm in the presence of wild type HIV-1 Gag. Wild-type and chimeric Gag proteins were capable of coassembly into a single VLP as viewed by correlative fluorescence SEM if, and only if, the CA domain was derived from the same virus. These results imply that the primary selectivity of Gag-Gag interactions is determined by the CA domain.

scholarly journals A Novel MVA-Based HIV Vaccine Candidate (MVA-gp145-GPN) Co-Expressing Clade C Membrane-Bound Trimeric gp145 Env and Gag-Induced Virus-Like Particles (VLPs) Triggered Broad and Multifunctional HIV-1-Specific T Cell and Antibody Responses

Viruses ◽  
2019 ◽  
Vol 11 (2) ◽  
pp. 160 ◽  
Author(s):  
Beatriz Perdiguero ◽  
Cristina Sánchez-Corzo ◽  
Carlos Sorzano ◽  
Lidia Saiz ◽  
Pilar Mediavilla ◽  
...  
Keyword(s):  
Neutralizing Antibodies ◽  
Vaccine Candidate ◽  
Hiv Vaccine ◽  
Replication Capacity ◽  
Virus Like Particles ◽  
Modified Vaccinia Virus Ankara ◽  
Clade C ◽  
Membrane Bound ◽  
Hiv 1

The development of an effective Human Immunodeficiency Virus (HIV) vaccine that is able to stimulate both the humoral and cellular HIV-1-specific immune responses remains a major priority challenge. In this study, we described the generation and preclinical evaluation of single and double modified vaccinia virus Ankara (MVA)-based candidates expressing the HIV-1 clade C membrane-bound gp145(ZM96) trimeric protein and/or the Gag(ZM96)-Pol-Nef(CN54) (GPN) polyprotein that was processed to form Gag-induced virus-like particles (VLPs). In vitro characterization of MVA recombinants revealed the stable integration of HIV-1 genes without affecting its replication capacity. In cells that were infected with Env-expressing viruses, the gp145 protein was inserted into the plasma membrane exposing critical epitopes that were recognized by broadly neutralizing antibodies (bNAbs), whereas Gag-induced VLPs were released from cells that were infected with GPN-expressing viruses. VLP particles as well as purified MVA virions contain Env and Gag visualized by immunoelectron microscopy and western-blot of fractions that were obtained after detergent treatments of purified virus particles. In BALB/c mice, homologous MVA-gp145-GPN prime/boost regimen induced broad and polyfunctional Env- and Gag-specific CD4 T cells and antigen-specific T follicular helper (Tfh) and Germinal Center (GC) B cells, which correlated with robust HIV-1-specific humoral responses. Overall, these results support the consideration of MVA-gp145-GPN vector as a potential vaccine candidate against HIV-1.

scholarly journals Assembly, structure, and antigenic properties of virus-like particles rich in HIV-1 envelope gp120

Virology ◽  
2004 ◽  
Vol 321 (1) ◽  
pp. 75-86 ◽  
Author(s):  
Ira Berkower ◽  
Megan Raymond ◽  
Jacqueline Muller ◽  
Angelo Spadaccini ◽  
Anne Aberdeen
Keyword(s):  
Virus Like Particles ◽  
Antigenic Properties ◽  
Assembly Structure ◽  
Hiv 1 ◽  
Envelope Gp120
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