Protein nanovaccine confers robust immunity against Toxoplasma

Bissati, Kamal El; Zhou, Ying; Paulillo, Sara M.; Raman, Senthil Kumar; Karch, Christopher P.; Roberts, Craig W.; Lanar, David E.; Reed, Steve; Fox, Chris; Carter, Darrick; Alexander, Jeff; Sette, Alessandro; Sidney, John; Lorenzi, Hernán; Begeman, Ian J.; Burkhard, Peter; McLeod, Rima

doi:10.1038/s41541-017-0024-6

Cited by 51 publications

(49 citation statements)

References 52 publications

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“…SAPNs are based on coiled-coil domains and are traditionally expressed in Escherichia coli 8 . SAPN vaccine candidates have been developed for a variety of diseases such as malaria, SARS, influenza, toxoplasmosis, and HIV-1 9,10,11,12,13,14,15,16,17,18,19 . The design of each SAPN candidate is specific to the pathogen of interest, however, the production, purification, and refolding techniques are generally broadly applicable.…”

Section: Introductionmentioning

confidence: 99%

Production of <em>E. coli</em>-expressed Self-Assembling Protein Nanoparticles for Vaccines Requiring Trimeric Epitope Presentation

Karch

Burkhard

Matyas

et al. 2019

JoVE

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Self-assembling protein nanoparticles (SAPNs) function as repetitive antigen displays and can be used to develop a wide range of vaccines for different infectious diseases. In this article we demonstrate a method to produce a SAPN core containing a six-helix bundle (SHB) assembly that is capable of presenting antigens in a trimeric conformation. We describe the expression of the SHB-SAPN in an E. coli system, as well as the necessary protein purification steps. We included an isopropanol wash step to reduce the residual bacterial lipopolysaccharide. As an indication of the protein identity and purity, the protein reacted with known monoclonal antibodies in Western blot analyses. After refolding, the size of the particles fell in the expected range (20 to 100 nm), which was confirmed by dynamic light scattering, nanoparticle tracking analysis, and transmission electron microscopy. The methodology described here is optimized for the SHB-SAPN, however, with only slight modifications it can be applied to other SAPN constructs. This method is also easily transferable to large scale production for GMP manufacturing for human vaccines. Video LinkThe video component of this article can be found at https://www.jove.com/video/60103/ 9 . This candidate was recognized by known monoclonal antibodies to HIV-1 envelope protein. Mice immunized with V1V2-SHB-SAPN raised V1V2 specific antibodies, that, most importantly, bound to gp70 V1V2, the correct conformational epitopes 9 . The SHB-SAPN core could have other functions beyond the role as a carrier for the HIV-1 V1V2 loop. Here we describe a detailed methodology for the expression, purification, refolding, and validation of the SHB-SAPN core. The sequence selection, nanoparticle design, the molecular cloning, and transformation of E. coli have been previously described 9 . Journal of Visualized Experiments August 2019 | 150 | e60103 | Page 2 of 11 Protocol 1. Expression of the SHB-SAPN Protein in E. coli BL21(DE3) 1. Mix 95 mL of component A and 5 mL of component B of the media in a 2 L sterile glass Erlenmeyer flask as per the manufacturer's instructions (see theTable of Materials). Add ampicillin to a final concentration of 100 μg/mL. 2. Inoculate the media with E. coli from a previously established glycerol stock culture. Incubate culture at 30 °C with shaking at 200 rotations per minute (rpm) for 48 h. NOTE: The used E. coli BL21 (DE3) stock contained the ampicillin resistant expression vector 23 with the SHB-SAPN gene. Although the general protocol of the media recommends 24 h of incubation at 37 °C, 48 h of incubation at 30 °C gave higher yield for SHB-SAPN. 3. Transfer the culture to two 50 mL conical tubes. Centrifuge the tubes at 4,000 x g for 10 min with a fixed angle rotor at 4 °C. Remove the supernatant and save the pellet to harvest cells. NOTE: The cell pellet can be either processed immediately or frozen at -80 °C until use. 2. Lysis of E. coli BL21(DE3) by sonication NOTE: Use nonpyrogenic plasticware and glassware baked at 250 °C for at least 30 min. Tris(2-car...

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Section: Introductionmentioning

confidence: 99%

Production of <em>E. coli</em>-expressed Self-Assembling Protein Nanoparticles for Vaccines Requiring Trimeric Epitope Presentation

Karch

Burkhard

Matyas

et al. 2019

JoVE

Self Cite

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“…Antigens and adjuvants can be inserted depending on the expected structure from in silico modeling [71] . Subunit malaria [72] , HIV [73] , toxoplasma [74] and severe acute respiratory syndrome [75] . Wahome et al, encapsulated two HIV protein epitopes (4E10 and 2F5) onto SAPN surface and observed enhanced production of epitope-specific neutralizing antibodies.…”

Section: Protein/peptide-based Nanovaccinesmentioning

confidence: 99%

Advancements in prophylactic and therapeutic nanovaccines

et al. 2020

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“…A promising alternative delivery system for subunitbased vaccines has been developed recently [134] and used with vaccine candidates against several infectious diseases such as HIV [142], toxoplasma [143][144][145], SARS [146], influenza [147] and/or malaria [148][149][150]. The technique is known as Self-Assembling Protein Nanoparticles (SAPNs) and involves the expression of a peptide/ protein containing a target antigen covalently linked to an adjuvant sequence (flagellin-derived) and, in some cases, a universal epitope such as the Pan-DR T-helper epitope (PADRE) sequence.…”

Section: Future Directionsmentioning

confidence: 99%

Plasmodium falciparum pre-erythrocytic stage vaccine development

Molina-Franky

Cuy-Chaparro

Camargo

et al. 2020

Malar J

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Worldwide strategies between 2010 and 2017 aimed at controlling malarial parasites (mainly Plasmodium falciparum) led to a reduction of just 18% regarding disease incidence rates. Many biologically-derived anti-malarial vaccine candidates have been developed to date; this has involved using many experimental animals, an immense amount of work and the investment of millions of dollars. This review provides an overview of the current state and the main results of clinical trials for sporozoite-targeting vaccines (i.e. the parasite stage infecting the liver) carried out by research groups in areas having variable malaria transmission rates. However, none has led to promising results regarding the effective control of the disease, thereby making it necessary to complement such efforts at finding/introducing new vaccine candidates by adopting a multi-epitope, multi-stage approach, based on minimal subunits of the main sporozoite proteins involved in the invasion of the liver.

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Protein nanovaccine confers robust immunity against Toxoplasma

Cited by 51 publications

References 52 publications

Production of <em>E. coli</em>-expressed Self-Assembling Protein Nanoparticles for Vaccines Requiring Trimeric Epitope Presentation

Production of <em>E. coli</em>-expressed Self-Assembling Protein Nanoparticles for Vaccines Requiring Trimeric Epitope Presentation

Advancements in prophylactic and therapeutic nanovaccines

Plasmodium falciparum pre-erythrocytic stage vaccine development

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