Name: production of e. coli-expressed self-assembling protein nanoparticles for vaccines requiring trimeric epitope presentation
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Description: Watch this Scientific Journal Video about Production of E. coli-expressed Self-Assembling Protein Nanoparticles for Vaccines Requiring Trimeric Epitope Presentation at JoVE.com

This work was supported by a cooperative agreement (W81XWH-11-2-0174) between the Henry M Jackson Foundation for the Advancement of Military Medicine, Inc., and the US Department of Defense. The anti-HIV-1 gp41 mAb 167-D IV antibody was received from Dr. Susan Zolla-Pazner through the NIH AIDS Reagent Program.

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The views expressed are those of the authors and should not be construed to represent the positions of the US Army or the Department of Defense. Peter Burkhard has an interest in the company called Alpha-O Peptides AG and has patents on the technology. The other authors have no affiliation or financial involvement with any company with a financial interest on the subject matter presented in this paper.

Nanotechnology provides many advantages and solutions for subunit vaccine development. Nanovaccines can repeatedly present antigens as particulates to the host immune system increasing immunogenicity26. While there are many different types of nanovaccines, we believe that ones composed of de novo designed protein seem to be the strongest approach for vaccine development1. They can be engineered without any sequence homology to the host proteins and present the antigen of interest in close to native-like conformation while providing low production cost and high product yields. A prime example of this approach is the SAPN technology, which we have applied to vaccines against multiple infectious diseases7. Addressing the difficulties in HIV-1 vaccine development, we have engineered a unique SHB-SAPN core to effectively present the V1V2 antigen in a native-like trimeric conformation9. Many vaccine targets, particularly for viral diseases, are present as trimers27. This phenomenon indicates that our SAPN design has wide implications for the development of subunit vaccines.
In this method, we demonstrate how to produce SHB-SAPNs in an E. coli expression system. We expressed high yields of protein (about 6 mg/100 mL of culture). The protein contained 10 histidines and was easily purified using an immobilized metal affinity chromatography with Ni2+ column. This length of the His-Tag was found to be the optimal for the highest protein yield. The purified protein contained the full-length of the designed protein as indicated by the presence of both the N-terminal HisTag and the C terminal heptad repeat. We utilized widely accepted techniques and optimized them for the expression, production, and characterization of the SHB-SAPN core. Lack of the production of the full-length protein during the development of a SAPN containing a new protein epitope could indicate an expression problem of the gene in the host cell. If it happens, the gene and the expression system must be redesigned and adapted to the described protocol. Modification of sonication time or intensity may also increase the concentration of the predicted full-length protein.
Refolded particles were in the expected size range (20 to 100 nm)8,24,25 as determined by DLS and nanoparticle tracking analysis. These results were further confirmed by using TEM. If there are problems in this step, it is normally due to a problem with the pH or ionic strength of the refolding buffer. When large size particles are detected on the particle sizing techniques, it indicates aggregation, which can be avoided by increasing the pH of the refolding buffer. If the particles are not detected by DLS, verify the concentration of the protein and check the pH of the buffer. The final protein concentration for DLS should be at least 100 &#181;g/mL. If the concentration is not the problem, it indicates the abundance of small, incompletely formed particles, whose concentration can be reduced by decreasing the pH. Alternatively, the sodium chloride concentration can be adjusted to the optimum range to minimize the presence of particles with unwanted size.
Finally, by using an isopropanol wash step during purification we were able to reduce contaminating LPS from the host E. coli to 0.01 EU/&#181;g of SAPN which is below the Food and Drug Administration (FDA) limit of 5 EU/kg of body weight for injectable products28. This level can be further reduced by using an anion exchange column also known as Q column. If high levels of endotoxin are still present, check all materials that were used for buffer preparation. Remember to use only depyrogenated glassware and endotoxin free plasticware in this method.
These results indicate that we have successfully developed a method to produce the SHB-SAPN core that can be used for pre-clinical immunization studies. This method with only slight modifications, if any, can be applied to the purification of SHB-SAPNs when an antigen of interest is added. Using this method as a starting point one of the major changes is in the elution step. Different proteins elute at different imidazole concentrations that must be determined experimentally. The other major difference might be the composition of the refolding buffer. Optimization would require testing different pH conditions as well as ionic strengths.
In consideration of future work, only two slight modifications are needed to allow human application of the SHB-SAPN. The first is that the expression vector needs to be changed to a kanamycin resistance selectable marker due to the ampicillin allergy in humans29. The other major requirement of the protein manufacturing for human use is to produce the SHB-SAPN in animal product-free media. A small-scale study already indicated a reasonable yield of protein in a plant-based media. The work presented here is easily scalable for ultimate GMP production as demonstrated with a malaria vaccine candidate, FMP01416. This large scale FMP014-SAPN production included both the anion exchange and the cation exchange steps to further reduce LPS and Ni2+ content from the final product. This bacterial-expressed SAPN has been already scaled up for an upcoming Phase 1/2a clinical trial.

While traditional vaccine development has focused on the inactivated or attenuated pathogens, the focus of modern vaccines has shifted toward subunit vaccines1. This approach can lead to a more targeted response, and potentially more efficacious vaccine candidates. However, one of the main drawbacks is that subunit vaccines are not particulates like whole organisms which can result in reduced immunogenicity2. A nanoparticle as a repetitive antigen display system can have the benefits of both the targeted subunit vaccine approach as well as the particulate nature of the whole organism1,3.
Among the existing types of nanovaccines, rationally designed protein assemblies allow for the design and development of vaccine candidates that can present multiple copies of the antigen potentially in a native-like conformation1,4,5,6. One example of these protein assemblies are the self-assembling protein nanoparticles (SAPNs)7. SAPNs are based on coiled-coil domains and are traditionally expressed in Escherichia coli8. SAPN vaccine candidates have been developed for a variety of diseases such as malaria, SARS, influenza, toxoplasmosis, and HIV-19,10,11,12,13,14,15,16,17,18,19. The design of each SAPN candidate is specific to the pathogen of interest, however, the production, purification, and refolding techniques are generally broadly applicable.
One of our current interests is an effective HIV-1 vaccine. In RV144&#8212;the only Phase III clinical trial of an HIV-1 vaccine that demonstrated modest efficacy&#8212;the reduced risk of infection was correlated with IgG antibodies to the V1V2 loop of the envelope protein20,21. The native-like trimeric presentation of this region is thought to be important for protective immunogenicity22. To present the V1V2 loop in as close to native-like conformation as possible, we developed a proof of principle SAPN vaccine candidate that contained the HIV-1 envelope post-fusion six-helix bundle (SHB) to present the V1V2 loop into the correct conformation9. 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 epitopes9. 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 described9.

<table><tbody><tr><td>10x Tris/Glycine/SDS</td><td>BioRad</td><td>1610732</td><td>1 L</td></tr><tr><td>2-Mercaptoethanol</td><td>BioRad</td><td>1610710</td><td>25 mL</td></tr><tr><td>2-propanol</td><td>Fisher</td><td>BP26181</td><td>4 L</td></tr><tr><td>2x Laemmli Sample Buffer</td><td>BioRad</td><td>1610737</td><td>30 mL</td></tr><tr><td>40ul Cuvette Pack of 100 with Stoppers</td><td>Malvern Panalytical</td><td>ZEN0040</td><td>100 pack</td></tr><tr><td>4&amp;ndash;20% Mini-PROTEAN TGX Precast Protein Gels, 10-well, 30 &amp;micro;l</td><td>BioRad</td><td>4561093</td><td>10 pack</td></tr><tr><td>Ampicillin</td><td>Fisher</td><td>BP1760-25</td><td>25 g</td></tr><tr><td>Anti-6X His tag antibody [HIS.H8]</td><td>AbCam</td><td>ab18184</td><td>100 mg</td></tr><tr><td>Anti-HIV-1 gp41 Monoclonal (167-D IV)</td><td>AIDS Reagent Repository</td><td>11681</td><td>100 mg</td></tr><tr><td>BCIP/NBT Substrate, Solution</td><td>Southern Biotech</td><td>0302-01 &amp;nbsp;</td><td>100 mL</td></tr><tr><td>Corning&amp;nbsp;Disposable Vacuum Filter/Storage Systems</td><td>Fisher</td><td>09-761-108</td><td>A variety of sizes</td></tr><tr><td>Formvar/Carbon 400 mesh, Copper approx. grid hole size: 42&amp;micro;m</td><td>Ted Pella, Inc</td><td>01754-F</td><td>25 pack</td></tr><tr><td>GE Healthcare&amp;nbsp;5 mL HisTrap HP Prepacked Columns</td><td>GE HealthCare</td><td>45-000-325</td><td>5 pack</td></tr><tr><td>Glycerol</td><td>Fisher</td><td>BP229-4</td><td>4 L</td></tr><tr><td>Goat Anti-Mouse IgG H&amp;amp;L (Alkaline Phosphatase)</td><td>ABCam</td><td>ab97020</td><td>1 mg</td></tr><tr><td>Imidazole</td><td>Fisher</td><td>O3196-500</td><td>500 g</td></tr><tr><td>Instant NonFat Dry Milk</td><td>Quality Biological</td><td>A614-1003</td><td>10 pack</td></tr><tr><td>Kinetic-QCL Kinetic Chormogenic LAL Assay</td><td>Lonza Walkersville</td><td>50650U</td><td>192 Test Kit</td></tr><tr><td>LAL Reagent Grade Multi-well Plates</td><td>Lonza Walkersville</td><td>25-340</td><td>1 plate</td></tr><tr><td>Magic Media&lt;em&gt; E. coli&lt;/em&gt; Expression Medium</td><td>ThermoFisher</td><td>K6803</td><td>1 L</td></tr><tr><td>MilliporeSigma Millex Sterile Syringe 0.22 mm Filters</td><td>Millipore</td><td>SLGV033RB</td><td>250 pack</td></tr><tr><td>Mouse Anti-Human IgG Fc-AP</td><td>Southern Biotech</td><td>9040-04 &amp;nbsp;</td><td>1.0 mL</td></tr><tr><td>One Shot BL21 Star (DE3) Chemically Competent &lt;em&gt;E. coli&lt;/em&gt;</td><td>ThermoFisher</td><td>C601003</td><td>20 vials</td></tr><tr><td>Precision Plus Protein Unstained Protein Standards, Strep-tagged recombinant,</td><td>BioRad</td><td>1610363</td><td>1 mL</td></tr><tr><td>Slide-A-Lyzer Dialysis Cassettes, 10K MWCO, 12 mL</td><td>ThermoFisher</td><td>66810</td><td>8 pack</td></tr><tr><td>Sodium Chloride</td><td>Fisher</td><td>BP358-212</td><td>2.5 kg</td></tr><tr><td>Sodium Phosphate Monobasic</td><td>Fisher</td><td>BP329-500</td><td>500 g</td></tr><tr><td>Tris Base</td><td>Fisher</td><td>BP152-1</td><td>1 kg</td></tr><tr><td>Tris-(2-carboxyethyl)phosphine hydrochloride</td><td>Biosynth International</td><td>C-1818</td><td>100 g</td></tr><tr><td>Uranyl Acetate, Reagent, A.C.S</td><td>Electron Micoscopy Services</td><td>541-09-3</td><td>25 g</td></tr><tr><td>Urea</td><td>Fisher</td><td>BP169-500</td><td>2.5 kg</td></tr><tr><td>Whatman qualitative filter paper</td><td>Sigma Aldrich</td><td>WHA10010155</td><td>pack of 500</td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;&amp;nbsp;Equipment&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>ChromLab Software ver 4</td><td>BioRad</td><td>12009390</td><td>Software</td></tr><tr><td>Epoch 2 Microplate Spectrophotometer</td><td>BioTek</td><td>EPOCH2</td><td>Plate Reader</td></tr><tr><td>Fiberlite F14-14 x 50cy Fixed-Angle Rotor</td><td>ThermoFisher</td><td>096-145075</td><td>Rotor</td></tr><tr><td>Gel Doc EZ Gel Documentation System</td><td>BioRad</td><td>1708270</td><td>Gel Imager for Stain free Gels</td></tr><tr><td>JEOL TEM</td><td>JEOL</td><td>1400</td><td>Transmission Electron Microscope</td></tr><tr><td>Mini-PROTEAN Tetra Vertical Electrophoresis Cell for Mini Precast Gels</td><td>BioRad</td><td>1658004</td><td>To run gels</td></tr><tr><td>NanoDrop One Microvolume UV-Vis Spectrophotometer</td><td>ThermoFisher</td><td>ND-ONE-W</td><td>For Protein Concentration</td></tr><tr><td>NanoSight NS300</td><td>Malvern Panalytical</td><td></td><td>Particle Sizing</td></tr><tr><td>NanoSight NTA software NTA</td><td>Malvern Panalytical</td><td></td><td>Particle Sizing</td></tr><tr><td>New Brunswick Innova 44/44R</td><td>Eppendorf</td><td>M1282-0000</td><td>Incubator/Shaker</td></tr><tr><td>NGC Quest 10 Chromatography System</td><td>BioRad</td><td>7880001</td><td>FPLC to aid in protein purification</td></tr><tr><td>PELCO easiGlow Glow Discharge Cleaning System</td><td>Ted Pella, INC</td><td>91000S</td><td>To clean grids</td></tr><tr><td>PowerPac Universal Power Supply</td><td>BioRad</td><td>1645070</td><td>To run gels</td></tr><tr><td>Rocker Shaker</td><td>Daigger</td><td>EF5536A</td><td>For Western</td></tr><tr><td>Sonifer 450</td><td>Branson</td><td>also known as 096-145075&amp;nbsp;</td><td>Sonicator</td></tr><tr><td>Thermo Scientific Sorvall LYNX 4000 Superspeed Centrifuge</td><td>ThermoFisher</td><td>75-006-580</td><td>Centrifuge</td></tr><tr><td>Trans-Blot Turbo Mini Nitrocellulose Transfer Packs</td><td>BioRad</td><td>1704158</td><td>For Western</td></tr><tr><td>Trans-Blot Turbo Transfer System</td><td>BioRad</td><td>1704150</td><td>For Western</td></tr><tr><td>Vortex-Genie 2</td><td>Daigger</td><td>EF3030A</td><td>Vortex</td></tr><tr><td>Zetasizer Nano ZS</td><td>Malvern Panalytical</td><td></td><td>Particle Sizing</td></tr><tr><td>Zetasizer Software</td><td>Malvern Panalytical</td><td></td><td>Particle Sizing</td></tr></tbody></table>

production of e. coli-expressed self-assembling protein nanoparticles for vaccines requiring trimeric epitope presentation

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.

The fully assembled SHB-SAPN shown here is built upon protein sequences (Figure 1A) that are predicted to fold into a particle that contains 60 copies of the monomer (Figure 1B). Figure 2 provides an outline of the method for the production, purification, and identification of the SHB-SAPN core. E. coli from a glycerol stock that contained a pPep-T expression vector with gene sequence of the SHB-SAPN core were induced in BL21 (DE3) E. coli. Bacterial cells were successfully grown and lysed under denaturing and reducing conditions.
Total cell lysate was used to purify SHB-SAPN monomers by FPLC using a Ni2+ column (Figure 3A). The FPLC chromatograph demonstrates that protein eluted both at 150 mM and 500 mM imidazole (Figure 3A). The chromatogram also shows two other peaks at 185 mL and 210 mL total volume corresponding to the isopropanol wash and the imidazole-free wash, respectively. The fractions and the purity of the recombinant protein were identified by gradient SDS-PAGE gels (Figure 3B). The protein of interest was primarily located in fractions 68&#8210;79 (278&#8210;300 mL total volume). These fractions were combined for further analyses. Western blot with anti-His antibody (N-terminal) and 167-D-IV antibody (C-terminus) indicated that the pooled fractions were indeed the protein of interest (Figure 4A,B). These blots also demonstrated the presence of the SHB-SAPN multimers. Earlier washes and elution fractions tended to contain a higher concentration of multimerized protein and were therefore excluded.
The samples that contained the protein monomers of interest were folded into the fully assembled SHB-SAPN by dialysis. Particle size distribution was determined by DLS and nanoparticle tracking analysis (Figure 5A,B). The DLS identified particles with a Z-average hydrodynamic diameter of 67 nm while the NTA system measured a mean size of 81 nm. The slight size differences were due to the particle sizing techniques, however the size from both analyses were in the expected range of 20&#8210;100 nm8,24,25. SHB-SAPNs were visualized by TEM and the images showed well-formed individual particles with the size distribution obtained from the two particle sizing techniques (Figure 5C).
During the purification of the protein, the column was washed with isopropanol to decrease the LPS contamination in the final SHB-SAPN product. To verify if the endotoxin level was acceptable for immunization, the concentration of LPS in SHB-SAPN samples purified with or without the isopropanol wash step was determined by a kinetic LAL assay. The results indicated that the isopropanol wash decreased the endotoxin levels from &#62;0.25 EU/&#181;g to 0.010 EU/&#181;g of SHB-SAPN protein (Table 1).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/60103/60103fig1.jpg" /> 
Figure 1: SHB-SAPN protein sequence and structure. (A) The amino acid sequence of the SHB-SAPN monomer. (B) Computer model of the structure of the fully assembled SHB-SAPN core consisting of 60 protein monomers. Color scheme for amino acid sequences: Grey = HisTag; Green = pentamer; Dark blue = de novo designed trimer; Light blue = six-helix bundle. <a href="https://jove.unicartagenaproxy.elogim.com/files/ftp_upload/60103/60103fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/60103/60103fig2.jpg" /> 
Figure 2: Flowchart of the protocol for SHB-SAPN production. Color scheme: Black = expression of the protein monomer in E. coli; Dark grey = monomer purification; Medium grey = monomer identification; Light grey = refolding and characterization; White = fully assembled SHB-SAPN product. In steps labeled with dark and medium grey, the protein is under denaturing and reducing conditions. <a href="https://jove.unicartagenaproxy.elogim.com/files/ftp_upload/60103/60103fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/60103/60103fig3.jpg" /> 
Figure 3: Protein purification of the SHB-SAPN monomers. (A) Chromatograph from the FPLC purification. Green line above the chromatogram indicates the purification steps. Blue line in the chromatogram represents the optical density of the fractions at 280 nm wavelength. The black line shows what percent of buffer B (8 M urea, 20 mM Tris, 50 mM sodium phosphate monobasic, 5 mM TCEP, 500 mM Imidazole, pH 8.5) that was used in each stage of the purification. (B) SDS-PAGE gels of the pooled fractions from the lysate (L), flow through (FT), first wash (W1), isopropanol wash (W2), third wash (W3), and individual fractions from the 150 mM imidazole (E150) and 500 mM imidazole (E500) elution steps of the purification. Molecular markers in the first lane (M) identify bands between 10 and 250 kDa. Target protein is indicated by a black arrow. <a href="https://jove.unicartagenaproxy.elogim.com/files/ftp_upload/60103/60103fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/60103/60103fig4.jpg" /> 
Figure 4: Identification of the protein by western blot. All lanes are loaded with 100 ng of protein. (A) Results of a western blot with anti-6x HisTag. (B) Results of a western blot with a 167-D-IV HIV-1 monoclonal antibody. Lanes are labeled as: M = molecular weight marker; 1 = lysate; 2 = Flow through; 3 = first wash; 4 = isopropanol wash (second wash); 5 = third wash; 6 = pooled volume fractions 56&#8210;61 (first elution peak), 7 = pooled volume factions 62&#8210;67 (between the two peaks), 8 = pooled volume fractions 68&#8210;78 (second elution peak). Target protein with the expected band size of 18.07 kDa as the monomeric SHB-SAPN band is indicated by a black arrow. Extra banding in lanes 7 and 8 are dimers, trimers, and multimers of the SHB-SAPN (red arrow). <a href="https://jove.unicartagenaproxy.elogim.com/files/ftp_upload/60103/60103fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/60103/60103fig5v2.jpg" /> 
Figure 5: Characterization of the refolded SHB-SAPN. (A). Particle size distribution as determined by DLS. (B) Particle size as determined by nanoparticle tracking (system). (C) Visualization of the SHB-SAPN particles by TEM. <a href="https://jove.unicartagenaproxy.elogim.com/files/ftp_upload/60103/60103fig5large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Sample</td>
			<td>Endotoxin (EU/mL)</td>
			<td>Endotoxin (EU/&#181;g of Protein)</td>
		</tr>
		<tr>
			<td>SAPN with Isopropanol Wash</td>
			<td>2.02</td>
			<td>0.01</td>
		</tr>
		<tr>
			<td>SAPN without Isopropanol Wash</td>
			<td>&#62;50</td>
			<td>&#62;0.25</td>
		</tr>
		<tr>
			<td>Negative Control</td>
			<td>Below detection level</td>
			<td>N/A</td>
		</tr>
	</tbody>
</table>
Table 1: Endotoxin levels of refolded SHB-SAPNs. Endotoxin levels in SHB-SAPN samples purified with or without an isopropanol wash presented both as endotoxin units/mL and endotoxin units/&#181;g of SHB-SAPN protein.

Watch this Scientific Journal Video about Production of E. coli-expressed Self-Assembling Protein Nanoparticles for Vaccines Requiring Trimeric Epitope Presentation at JoVE.com

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

A detailed method is provided here describing the purification, refolding, and characterization of self-assembling protein nanoparticles (SAPNs) for use in vaccine development.

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

Self-assembling protein nanoparticles (SAPNs) function as repetitive antigen displays and can be used to develop a wide range of vaccines for different ...

production-e-coli-expressed-self-assembling-protein-nanoparticles-for

Universidad de Cartagena UDEC

Research

JoVE Journal

Bioengineering

7.4K Views.  Walter Reed Army Institute of Research. A detailed method is provided here describing the purification, refolding, and characterization of self-assembling protein nanoparticles (SAPNs) for use in vaccine development.

Video: Production of E. coli-expressed Self-Assembling Protein Nanoparticles for Vaccines Requiring Trimeric Epitope Presentation

Homogeneous Glycoconjugate Produced by Combined Unnatural Amino Acid Incorporation and Click-Chemistry for Vaccine Purposes

Genetic code expansion is a powerful tool to introduce unnatural amino acids (UAAs) into proteins to modify their characteristics, to study or create new protein functions or to have access to protein conjugates. Stop codon suppression, in particular amber codon suppression, has emerged as the most popular method to genetically introduce UAAs at defined positions. This methodology is herein applied to the preparation of a carrier protein containing an UAA harboring a bioorthogonal functional group. This reactive handle can next be used to specifically and efficiently graft a synthetic oligosaccharide hapten to provide a homogeneous glycoconjugate vaccine. The protocol is limited to the synthesis of glycoconjugates in a 1:1 carbohydrate hapten/carrier protein ratio but amenable to numerous pairs of biorthogonal functional groups. Glycococonjugate vaccine homogeneity is an important criterion to ensure complete physico-chemical characterization, thereby, satisfying more and more demanding drug regulatory agency recommendations, a criterion which is unmet by classical conjugation strategies. Moreover, this protocol makes it possible to finely tune the structure of the actual conjugate vaccine, giving rise to tools to address structure-immunogenicity relationships.

Genetic code expansion is applied for the introduction of an unnatural amino acid bearing a biorthogonal functional group on a carrier protein at a defined site. The biorthogonal function is further used for the site-selective coupling of a carbohydrate antigen to provide a homogeneous glycoconjugate vaccine.

Genetic code expansion is a powerful tool to introduce unnatural amino acids (UAAs) into proteins to modify their characteristics, to study or create new ...

Synthesis and Characterization of mRNA-Loaded Poly(Beta Aminoesters) Nanoparticles for Vaccination Purposes

Vaccination has been one of the major successes of modern society and is indispensable in controlling and preventing disease. Traditional vaccines were composed of entire or fractions of the infectious agent. However, challenges remain, and new vaccine technologies are mandatory. In this context, the use of mRNA for immunizing purposes has shown an enhanced performance, as demonstrated by the speedy approval of two mRNA vaccines preventing SARS-CoV-2 infection. Beyond success in preventing viral infections, mRNA vaccines can also be used for therapeutic cancer applications.
Nevertheless, the instability of mRNA and its fast clearance from the body due to the presence of nucleases makes its naked delivery not possible. In this context, nanomedicines, and specifically polymeric nanoparticles, are critical mRNA delivery systems. Thus, the aim of this article is to describe the protocol for the formulation and test of an mRNA vaccine candidate based on the proprietary polymeric nanoparticles. The synthesis and chemical characterization of the poly(beta aminoesters) polymers used, their complexation with mRNA to form nanoparticles, and their lyophilization methodology will be discussed here. This is a crucial step for decreasing storage and distribution costs. Finally, the required tests to demonstrate their capacity to in vitro transfect and mature model dendritic cells will be indicated. This protocol will benefit the scientific community working on vaccination because of its high versatility that enables these vaccines to prevent or cure a wide variety of diseases.

Synthesis and Characterization of mRNA-Loaded PolyBeta Aminoesters Nanoparticles for Vaccination Purposes

Here, a simple protocol is presented for producing mRNA nanoparticles based on poly(beta aminoester) polymers, easy to be tailored by changing the encapsulated mRNA. The workflow for synthesizing the polymers, the nanoparticles, and their in vitro essential characterization are also described. A proof-of-concept regarding immunization is also added.

Vaccination has been one of the major successes of modern society and is indispensable in controlling and preventing disease. Traditional vaccines were ...

A "Plug-And-Display" Nanoparticle Vaccine Platform Based on Outer Membrane Vesicles Displaying SARS-CoV-2 Receptor-Binding Domain

Biomimetic nanoparticles obtained from bacteria or viruses have attracted substantial interest in vaccine research and development. Outer membrane vesicles (OMVs) are mainly secreted by gram-negative bacteria during average growth, with a nano-sized diameter and self-adjuvant activity, which may be ideal for vaccine delivery. OMVs have functioned as a multifaceted delivery system for proteins, nucleic acids, and small molecules. To take full advantage of the biological characteristics of OMVs, bioengineered Escherichia coli-derived&#160;OMVs were utilized as a carrier and SARS-CoV-2 receptor-binding domain (RBD) as an antigen to construct a &#34;Plug-and-Display&#34; vaccine platform. The SpyCatcher (SC) and SpyTag (ST) domains in Streptococcus pyogenes were applied to conjugate OMVs and RBD. The Cytolysin A (ClyA) gene was translated with the SC gene as a fusion protein after plasmid transfection, leaving a reactive site on the surface of the OMVs. After mixing RBD-ST in a conventional buffer system overnight, covalent binding was formed between the OMVs and RBD. Thus, a multivalent-displaying OMV vaccine was achieved. By replacing with diverse antigens, the OMVs vaccine platform can efficiently display a variety of heterogeneous antigens, thereby potentially rapidly preventing infectious disease epidemics. This protocol describes a precise method for constructing the OMV vaccine platform, including production, purification, bioconjugation, and characterization.

The present protocol describes the bioengineering of outer membrane vesicles to be a "Plug-and-Display" vaccine platform, including production, purification, bioconjugation, and characterization.

Biomimetic nanoparticles obtained from bacteria or viruses have attracted substantial interest in vaccine research and development. Outer membrane ...

Cell-Free Scaled Production and Adjuvant Addition to a Recombinant Major Outer Membrane Protein from Chlamydia muridarum for Vaccine Development

Subunit vaccines offer advantages over more traditional inactivated or attenuated whole-cell-derived vaccines in safety, stability, and standard manufacturing. To achieve an effective protein-based subunit vaccine, the protein antigen often needs to adopt a native-like conformation. This is particularly important for pathogen-surface antigens that are membrane-bound proteins. Cell-free methods have been successfully used to produce correctly folded functional membrane protein through the co-translation of nanolipoprotein particles (NLPs), commonly known as nanodiscs. 
This strategy can be used to produce subunit vaccines consisting of membrane proteins in a lipid-bound environment. However, cell-free protein production is often limited to small scale (&lt;1 mL). The amount of protein produced in small-scale production runs is usually sufficient for biochemical and biophysical studies. However, the cell-free process needs to be scaled up, optimized, and carefully tested to obtain enough protein for vaccine studies in animal models. Other processes involved in vaccine production, such as purification, adjuvant addition, and lyophilization, need to be optimized in parallel. This paper reports the development of a scaled-up protocol to express, purify, and formulate a membrane-bound protein subunit vaccine. 
Scaled-up cell-free reactions require optimization of plasmid concentrations and ratios when using multiple plasmid expression vectors, lipid selection, and adjuvant addition for high-level production of formulated nanolipoprotein particles. The method is demonstrated here with the expression of a chlamydial major outer membrane protein (MOMP) but may be widely applied to other membrane protein antigens. Antigen effectiveness can be evaluated in vivo through immunization studies to measure antibody production, as demonstrated here.

Cell-Free Scaled Production and Adjuvant Addition to a Recombinant Major Outer Membrane Protein from Chlamydia muridarum for Vaccine Development

This protocol describes using commercial, cell-free protein expression kits to produce membrane proteins supported in nanodisc that can be used as antigens in subunit vaccines.

Subunit vaccines offer advantages over more traditional inactivated or attenuated whole-cell-derived vaccines in safety, stability, and standard ...

Immunology and Infection

Detection of Neutralization-sensitive Epitopes in Antigens Displayed on Virus-Like Particle (VLP)-Based Vaccines Using a Capture Assay

The virus-like particle (VLP) capture assay is an immunoprecipitation method, commonly known as a &#39;pull-down assay&#39; used to purify and isolate antigen-displaying VLPs. Surface antigen-specific antibodies are coupled to, and thus immobilized on a solid and insoluble matrix such as beads. Due to their high affinity to the target antigen, these antibodies can capture VLPs decorated with the cognate antigen anchored in the membrane envelope of the VLPs. This protocol describes the binding of antigen-specific antibodies to protein A- or G-conjugated magnetic beads. In our study, human immunodeficiency virus (HIV)-derived VLPs formed by the group-specific antigen (Gag) viral core precursor protein p55 Gag and displaying the envelope glycoproteins (Env) of HIV are examined. The VLPs are captured utilizing broadly neutralizing antibodies (bNAbs) directed against neutralization-sensitive epitopes in Env. The VLP capture assay outlined here represents a sensitive and easy-to-perform method to demonstrate that (i) the VLPs are decorated with the respective target antigen, (ii) the surface antigen retained its structural integrity as demonstrated by the epitope-specific binding of bNAbs used in the assay and (iii) the structural integrity of the VLPs revealed by the detection of Gag proteins in a subsequent Western blot-analysis. Consequently, the utilization of bNAbs for immunoprecipitation facilitates a prediction of whether VLP vaccines will be able to elicit a neutralizing B cell response in vaccinated humans. We anticipate that this protocol will furnish other researchers with a valuable and straightforward experimental approach to examine potential VLP-based vaccines.

Detection of Neutralization-sensitive Epitopes in Antigens Displayed on Virus-Like Particle VLP-Based Vaccines Using a Capture Assay

Here, we present a protocol to detect neutralization epitopes on antigen-displaying virus-like particles (VLPs). Immunoprecipitation of the human immunodeficiency virus (HIV)-derived VLPs is performed using envelope glycoproteins-specific monoclonal antibodies coupled to protein G-conjugated magnetic beads. Captured VLPs are subsequently subjected to SDS-PAGE and Western blot-analysis employing viral core protein Gag-specific antibodies.

The virus-like particle (VLP) capture assay is an immunoprecipitation method, commonly known as a &#39;pull-down assay&#39; used to purify and isolate ...

Preparation, Characteristics, Toxicity, and Efficacy Evaluation of the Nasal Self-Assembled Nanoemulsion Tumor Vaccine In Vitro and In Vivo

Epitope peptides have attracted widespread attention in the field of tumor vaccines because of their safety, high specificity, and convenient production; in particular, some MHC I-restricted epitopes can induce effective cytotoxic T lymphocyte activity to clear tumor cells. Additionally, nasal administration is an effective and safe delivery technique for tumor vaccines due to its convenience and improved patient compliance. However, epitope peptides are unsuitable for nasal delivery because of their poor immunogenicity and lack of delivery efficiency. Nanoemulsions (NEs) are thermodynamically stable systems that can be loaded with antigens and delivered directly to the nasal mucosal surface. Ile-Lys-Val-Ala-Val (IKVAV) is the core pentapeptide of laminin, an integrin-binding peptide expressed by human respiratory epithelial cells. In this study, an intranasal self-assembled epitope peptide NE tumor vaccine containing the synthetic peptide IKVAV-OVA257-264 (I-OVA) was prepared by a low-energy emulsification method. The combination of IKVAV and OVA257-264 can enhance antigen uptake by nasal mucosal epithelial cells. Here, we establish a protocol to study the physicochemical characteristics by transmission electron microscopy (TEM), atomic force microscopy (AFM), and dynamic light scattering (DLS); stability in the presence of mucin protein; toxicity by examining the cell viability of BEAS-2B cells and the nasal and lung tissues of C57BL/6 mice; cellular uptake by confocal laser scanning microscopy (CLSM); release profiles by imaging small animals in vivo; and the protective and therapeutic effect of the vaccine by using an E.G7 tumor-bearing model. We anticipate that the protocol will provide technical and theoretical clues for the future development of novel T cell epitope peptide mucosal vaccines.

Preparation, Characteristics, Toxicity, and Efficacy Evaluation of the Nasal Self-Assembled Nanoemulsion Tumor Vaccine In Vitro and In Vivo

Here, we present detailed methods for the preparation and evaluation of the nasal self-assembled nanoemulsion tumor vaccine in vitro and in vivo.

Epitope peptides have attracted widespread attention in the field of tumor vaccines because of their safety, high specificity, and convenient production; ...

Expression and Purification of Virus-like Particles for Vaccination

Virus-like particles (VLPs) and subviral particles (SVPs) are an alternative approach to viral vaccine design that offers the advantages of increased biosafety and stability over use of live pathogens. Non-infectious and self-assembling, VLPs are used to present structural proteins as immunogens, bypassing the need for live pathogens or recombinant viral vectors for antigen delivery. In this article, we demonstrate the different stages of VLP design and development for future applications in preclinical animal testing. The procedure includes the following stages: selection of antigen, expression of antigen in cell line of choice, purification of VLPs/SVPs, and quantification for antigen dosing. We demonstrate use of both mammalian and insect cell lines for expression of our antigens and demonstrate how methodologies differ in yield. The methodology presented may apply to a variety of pathogens and can be achieved by substituting the antigens with immunogenic structural proteins of the user&#39;s microorganism of interest. VLPs and SVPs assist with antigen characterization and selection of the best vaccine candidates.

Here, we present a protocol for synthesizing virus-like particles using either baculovirus or mammalian expression systems, and ultracentrifugation purification. This highly customizable approach is used to identify viral antigens as vaccine targets in a safe and flexible manner.

Virus-like particles (VLPs) and subviral particles (SVPs) are an alternative approach to viral vaccine design that offers the advantages of increased ...

Biology

Production and Imaging of Recombinant Protein-Filled Vesicles from E. coli

Optimized Production and Analysis of Recombinant Protein-Filled Vesicles from E. coli

This innovative system, using a short peptide tag, that exports multiple recombinant proteins in membrane bound vesicles from E. coli, provides an effective solution to a range of problems associated with bacterial recombinant protein expression. These recombinant vesicles compartmentalise proteins within a micro-environment that facilitates the production of otherwise challenging, toxic, insoluble, or disulfide-bond containing proteins from bacteria. Protein yield is increased considerably when compared to typical bacterial expression in the absence of the vesicle-nucleating peptide tag. The release of vesicle-packaged proteins supports isolation from the culture medium&#160;and permits long-term active protein storage. This technology gives rise to increased yields of vesicle-packaged, functional proteins for simplified downstream processing for a diverse range of applications from applied biotechnology to discovery science and medicine. In the present article and the associated video, a detailed protocol of the method is provided, which highlights key steps in the methodology to maximize recombinant protein-filled vesicle production.

Author Spotlight: The Production of Recombinant Proteins 

The present protocol describes a detailed method for the bacterial production of recombinant proteins, including typically insoluble or disulfide-bond containing proteins, packaged inside extracellular membrane-bound vesicles. This has the potential to be applied to versatile areas of scientific research, including applied biotechnology and medicine.

This innovative system, using a short peptide tag, that exports multiple recombinant proteins in membrane bound vesicles from E. coli, provides an ...

Generating a Multivalent-Displaying Outer Membrane Vesicle Vaccine

Source: Feng, R., et al. <a href="https://appjove.unicartagenaproxy.elogim.com/v/64213">A &#34;Plug-And-Display&#34; Nanoparticle Vaccine Platform Based on Outer Membrane Vesicles Displaying SARS-CoV-2 Receptor-Binding Domain.</a> J. Vis. Exp. (2022).
This video demonstrates a method to generate multivalent-displaying outer membrane vesicle (OMV) vaccines. Mammalian cells are transfected to express and secrete viral antigens fused to a SpyTag and a polyhistidine tag. These antigens are then purified using a nickel-nitrilotriacetic acid (Ni-NTA) affinity column. The purified antigen is mixed with outer membrane vesicles expressing surface-bound SpyCatcher (SC) protein. Multiple antigens covalently attach to SpyCatchers via their SpyTags (ST), creating OMVs that display the antigen, serving as a multivalent vaccine.

1. Plasmid construction

	Insert DNA encoding SpyCatcher sequence (Figure 1) into an ampicillin-resistant pThioHisA-Cytolysin A (ClyA) plasmid (see Table ...

JoVE Encyclopedia of Experiments

Immunology

Immunotherapy

Immunogenics and Vaccine Development

Purification of Self-Assembling Protein Nanoparticles using Affinity Chromatography

Source: Karch, C. P. et al., <a href="https://appjove.unicartagenaproxy.elogim.com/v/60103">Production of E. coli-expressed Self-Assembling Protein Nanoparticles for Vaccines Requiring Trimeric Epitope Presentation.</a>&#160;J. Vis. Exp. (2019)
This video showcases the purification of histidine-tagged self-assembled protein nanoparticles or SAPNs using affinity liquid chromatography. The resulting purified SAPN fractions are suitable for vaccine development, holding promise for potential immunotherapeutic applications.

1. Expression of the Self-assembling protein nanoparticles core containing a six-helix bundle (SHB-SAPN) Protein in E. coli BL21(DE3)

	Mix 95 mL of ...

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

Summary

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Chapters in this video

Homogeneous Glycoconjugate Produced by Combined Unnatural Amino Acid Incorporation and Click-Chemistry for Vaccine Purposes

Synthesis and Characterization of mRNA-Loaded Poly(Beta Aminoesters) Nanoparticles for Vaccination Purposes

A "Plug-And-Display" Nanoparticle Vaccine Platform Based on Outer Membrane Vesicles Displaying SARS-CoV-2 Receptor-Binding Domain

Cell-Free Scaled Production and Adjuvant Addition to a Recombinant Major Outer Membrane Protein from Chlamydia muridarum for Vaccine Development

Detection of Neutralization-sensitive Epitopes in Antigens Displayed on Virus-Like Particle (VLP)-Based Vaccines Using a Capture Assay

Preparation, Characteristics, Toxicity, and Efficacy Evaluation of the Nasal Self-Assembled Nanoemulsion Tumor Vaccine In Vitro and In Vivo

Expression and Purification of Virus-like Particles for Vaccination

Optimized Production and Analysis of Recombinant Protein-Filled Vesicles from E. coli

Generating a Multivalent-Displaying Outer Membrane Vesicle Vaccine

Purification of Self-Assembling Protein Nanoparticles using Affinity Chromatography