We wish to acknowledge the prior members of the Ross lab who have helped optimize the protocol for maximal efficiency and yields.

1. Mammalian Expression System for Generation of Influenza H3N2 VLP
<ol>
	<li>Subclone viral structural glycoproteins hemagglutinin (HA), neuraminidase (NA), and human immunodeficiency virus (HIV) Gag p55 into eukaryotic expression vectors, such as previously described pTR600.7</li>
	<li>Amplify DNA in chemically competent Escherichia coli (e.g., DH5&#945;) and isolate transfection-grade plasmid using a plasmid purification kit as per manufacturer&#39;s instructions. Amount of DNA is dependent on yield and user&#39;s needs.</li>
	<li>Maintain mammalian cell line, 293T, in growth media containing 10% fetal-bovine serum (FBS) at 37 &#176;C with 5% CO2 in a humidified incubator.</li>
	<li>Grow cells such that a T-150 tissue culture flask contains 45-75 x 106 cells per flask such that flask obtains complete cell adherence and 85-95% cell confluency by the following day. Inspect cells under the microscope for desired confluency. 
	Note: High cell density is typically recommended to yield optimal protein expression with minimal cytotoxicity when using commercial transfection reagents however over-confluency may result in accumulation of cellular waste by-products and reduce cell viability.</li>
	<li>On the day of transfection, prepare liposome and DNA mixture as per the manufacturer&#39;s recommendations and transfect DNA cells with a DNA composition of 1:1:2 of HA:NA:Gag with a total DNA quantity of 40 &#181;g per T-150 flask. Dilute DNA and liposome solutions in serum-free transfection media without antibiotics such that each flask contains a total volume of 20-30 ml. 
	Note: Ratio of gene constructs may require user optimization. Although not demonstrated here, a similar transfection procedure has been performed with respiratory syncytial virus (RSV) F and HIV Gag at a 1:1 ratio. For DENV and CHIKV single expression plasmids, a total of 20 &#181;g of DNA per T-150 flask are used for optimal expression. DNA:transfection reagent ratios are user optimized. Use recommended transfection medium based on transfection method, i.e., polycationic lipid transfections recommend reduction or absence of fetal bovine serum thus media may be supplemented with growth hormones and trace elements to support growth in the absence of serum.</li>
	<li>Return flasks to 37 &#176;C with 5% CO2 incubator. For a volume of 200 ml, use 9 T-150 flasks. The cells are maintained in transfection culture medium until the day of VLP harvest.</li>
	<li>Harvest culture supernatant from cells after 72-96 hr post-transfection depending on antigen, or when cell viability has decreased to 70-80%, as estimated by microscopic inspection. Transfer supernatant into 50 ml conical tubes and spin the cells down at 500 &#215; g for 5 min at 4 &#176;C to pellet cellular debris. 
	Note: Replacement of 3 day supernatant with fresh pre-warmed, serum-free transfection media to adherent cells will yield a second lot of VLPs with similar or slightly reduced yield and should be optimized by user.</li>
	<li>Pool supernatant and filter through a 0.22 &#181;m pore membrane before sedimentation via ultracentrifugation.</li>
	<li>Test the hemagglutination activity of the HA-expressing VLPs by standard hemagglutination assay14 using 0.8% turkey or mammalian red blood cells or proceed with antigen-specific ELISA. See Figure 2 for representative results.</li>
</ol>
2. CHIK VLP Expression Using Baculovirus/Insect Cell System
<ol>
	<li>Generate recombinant baculovirus expressing chikungunya virus capsid and envelope proteins (C-E3-E2-6K-E1) from S-27 strain using a commercial baculovirus expression system.</li>
	<li>Culture Spodoptera frugiperda Sf9 cells in serum-free Sf9 growth medium with 100 U/ml penicillin and 100 &#181;g/ml streptomycin at 28 &#176;C. Use 3-5 x 105 c/ml to initiate spinner flask cultures for suspension cell growth. Passage routinely when they reach cell densities of 2-4 x 106 c/ml (every 3-4 days).</li>
	<li>Culture Sf9 cells in suspension in spinner flasks with stirring at 130 rpm on a multipoint stirrer plate system. For proper aeration, maintain culture volumes at no more than half the volume of the spinner flask.</li>
	<li>For expression of CHIK VLPs, infect Sf9 cells at a density of 2 x 106 cells/ml with recombinant baculovirus at a multiplicity of infection of 1 and return to 28 &#176;C incubator. Typically, infect one or two spinner flasks, containing 250 ml Sf9 cells. 
	Note: While we do not use this method, Sf9 cells may be cultured in shaker flasks at 28 &#176;C using a shaker platform.</li>
	<li>Harvest cultures after 72-96 hr post-infection, or when cell viability has decreased to 70-80% as determined by Trypan Blue Exclusion according to manufacturer&#39;s protocol. 
	Note: The cells continue to proliferate while infected and there is a ~80% viability in Sf9 cultures infected with recombinant CHIK VLP baculovirus at 3 days post-infection (dpi). Baculovirus-infected cells are larger in appearance. Morphology may also change from round to oblong. In late-stages of baculovirus infections, the cells began to lyse.</li>
	<li>Transfer cultures directly from suspension culture into 50 ml conical tubes and spin the cells down at 500 x g for 5 min at 4 &#176;C.</li>
	<li>Collect supernatants and filter through a 0.22 &#181;m pore membrane before sedimentation via ultracentrifugation.</li>
</ol>
3. Sedimentation/Purification of VLP/SVPs
<ol>
	<li>Sterilize 25 mm x 89 mm open-top ultracentrifuge tubes with 70% ethanol in biosafety hood. Ensure the ethanol has dried off before use.</li>
	<li>Load up to 32 ml of supernatant into a clean tube. A minimal volume of 25 ml is recommended to prevent collapse and damage of inadequately filled ultracentrifuge tubes.</li>
	<li>Carefully underlay supernatants with sterile 3 ml 20% glycerol in PBS (v/v). Make sure tubes are balanced.</li>
	<li>Spin at 135,000 x g for 4 hr at 4 &#176;C.</li>
	<li>Aspirate supernatant, ensuring the pellet does not dislodge from tube.</li>
	<li>Resuspend sedimented VLPs at bottom of the tubes with sterile PBS by vigorously pipetting up and down. The amount of PBS needed to resuspend VLPs depends on VLP total protein yield and downstream applications. Typically, resuspend each VLP pellet in at least 100 &#181;l.</li>
	<li>Store samples 4 &#176;C for short-term storage and -80 &#176;C storage for long-term storage.</li>
</ol>
4. Determining Protein Yields and Specific Antigen Yields
<ol>
	<li>Determine the total protein content using a commercial protein quantification method, such as BCA assay as per manufacturer&#39;s protocol.</li>
	<li>To assess specific antigen content, perform direct enzyme-linked immunosorbent assay (ELISA) by coating serial dilutions of standard antigen and 2-5 &#181;g of total protein sample on to an ELISA 96-well flat bottom plate.
	<ol>
		<li>Follow with antigen-specific antibodies to probe the presence of the antigens on the plate and use conventional ELISA development substrates to produce detectable absorbance for microplate reader. See Figure 2 for representative results of HA and ELISA assay for a sample HA-expressing VLP; many combinations of HA and NA are possibly but yields may differ upon HA-NA compatibility15. 
		Note: Antigen-specific antibodies have variable affinities and it is recommended that endpoint-dilution titration of antibodies be determined before performing VLP quantification. Commercial antibodies will have suggested concentration or dilution ranges for use in ELISA, as well as suggested protocols.</li>
	</ol>
	</li>
</ol>

<ol>
	<li>Zeltins, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Construction+and+characterization+of+virus-like+particles:+a+review.">Construction and characterization of virus-like particles: a review.</a> Mol Biotechnol. 53, 92-107 (2013).</li><li>Jain, N. K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Formulation+and+stabilization+of+recombinant+protein+based+virus-like+particle+vaccines.">Formulation and stabilization of recombinant protein based virus-like particle vaccines.</a> Adv Drug Deliv Rev. , (2014).</li><li>Mair, C. M., Ludwig, K., Herrmann, A., Sieben, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Receptor+binding+and+pH+stability+-+how+influenza+A+virus+hemagglutinin+affects+host-specific+virus+infection.">Receptor binding and pH stability - how influenza A virus hemagglutinin affects host-specific virus infection.</a> Biochim. Biophys. Acta. 1838, 1153-1168 (2014).</li><li>. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Engerix-B+package+insert.">Engerix-B package insert.</a> GlaxoSmithKline, Research Triangle Park, NC. , (2013).</li><li>Giles, B. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+computationally+optimized+hemagglutinin+virus-like+particle+vaccine+elicits+broadly+reactive+antibodies+that+protect+nonhuman+primates+from+H5N1+infection.">A computationally optimized hemagglutinin virus-like particle vaccine elicits broadly reactive antibodies that protect nonhuman primates from H5N1 infection.</a> J Infect Dis. 205, 1562-1570 (2012).</li><li>Giles, B. M., Ross, T. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+computationally+optimized+broadly+reactive+antigen+(COBRA)+based+H5N1+VLP+vaccine+elicits+broadly+reactive+antibodies+in+mice+and+ferrets.">A computationally optimized broadly reactive antigen (COBRA) based H5N1 VLP vaccine elicits broadly reactive antibodies in mice and ferrets.</a> Vaccine. 29, 3043-3054 (2011).</li><li>Green, T. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=C3d+enhancement+of+neutralizing+antibodies+to+measles+hemagglutinin.">C3d enhancement of neutralizing antibodies to measles hemagglutinin.</a> Vaccine. 20, 242-248 (2001).</li><li>Bright, R. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cross-Clade+Protective+Immune+Responses+to+Influenza+Viruses+with+H5N1+HA+and+NA+Elicited+by+an+Influenza+Virus-Like+Particle.">Cross-Clade Protective Immune Responses to Influenza Viruses with H5N1 HA and NA Elicited by an Influenza Virus-Like Particle.</a> PLoS ONE. 3, e1501 (2008).</li><li>Yondola, M. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Budding+Capability+of+the+Influenza+Virus+Neuraminidase+Can+Be+Modulated+by+Tetherin.">Budding Capability of the Influenza Virus Neuraminidase Can Be Modulated by Tetherin.</a> J Virol. 85, 2480-2491 (2011).</li><li>Schneider-Ohrum, K., Ross, T. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Virus-like+particles+for+antigen+delivery+at+mucosal+surfaces.">Virus-like particles for antigen delivery at mucosal surfaces.</a> Curr Top Microbiol Immunol. 354, 53-73 (2012).</li><li>Murawski, M. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Newcastle+disease+virus-like+particles+containing+respiratory+syncytial+virus+G+protein+induced+protection+in+BALB/c+mice,+with+no+evidence+of+immunopathology.">Newcastle disease virus-like particles containing respiratory syncytial virus G protein induced protection in BALB/c mice, with no evidence of immunopathology.</a> J Virol. 84, 1110-1123 (2010).</li><li>Quan, F. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Viruslike+particle+vaccine+induces+protection+against+respiratory+syncytial+virus+infection+in+mice.">Viruslike particle vaccine induces protection against respiratory syncytial virus infection in mice.</a> J Infect Dis. 204, 987-995 (2011).</li><li>Ross, T. M., Tang, X., Lu, H. R., Olagnier, D., Kirchenbaum, G. A., Evans, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=COBRA-Based+Dengue+Tetravalent+Vaccine+Elicits+Neutralizing+Antibodies+Against+All+Four+Dengue+Serotypes.">COBRA-Based Dengue Tetravalent Vaccine Elicits Neutralizing Antibodies Against All Four Dengue Serotypes.</a> J Vaccine Immunotechnology. 1 (7), (2014).</li><li>. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Manual+for+the+Laboratory+Diagnosis+and+Virological+Surveillance+of+Influenza.">Manual for the Laboratory Diagnosis and Virological Surveillance of Influenza.</a> World Health Organization. , (2011).</li><li>Mitnaul, L. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Balanced+Hemagglutinin+and+Neuraminidase+Activities+Are+Critical+for+Efficient+Replication+of+Influenza+A+Virus.">Balanced Hemagglutinin and Neuraminidase Activities Are Critical for Efficient Replication of Influenza A Virus.</a> J Virol. 74, 6015-6020 (2000).</li><li>Jarvis, D. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Methods in Enzymology. 463, 191-222 (2009).</li><li>Pijlman, G. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enveloped+virus-like+particles+as+vaccines+against+pathogenic+arboviruses.">Enveloped virus-like particles as vaccines against pathogenic arboviruses.</a> Biotechnol J. 10, 659-670 (2015).</li><li>Park, J. K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protective+efficacy+of+crude+virus-like+particle+vaccine+against+HPAI+H5N1+in+chickens+and+its+application+on+DIVA+strategy.">Protective efficacy of crude virus-like particle vaccine against HPAI H5N1 in chickens and its application on DIVA strategy.</a> Influenza Other Respir Viruses. 7, 340-348 (2013).</li><li>Gangadharan, D., Smith, J., Weyant, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biosafety+Recommendations+for+Work+with+Influenza+Viruses+Containing+a+Hemagglutinin+from+the+A/goose/Guangdong/1/96+Lineage.+MMWR.+Recommendations+and+reports+:+Morbidity+and+mortality+weekly+report.">Biosafety Recommendations for Work with Influenza Viruses Containing a Hemagglutinin from the A/goose/Guangdong/1/96 Lineage. MMWR. Recommendations and reports : Morbidity and mortality weekly report.</a> Recommendations and reports / Centers for Disease Control. 62, 1-7 (2013).</li></ol>

Authors have nothing to disclose.

We have used the techniques described above to successfully express and purify SVPs and VLPs composed of multiple structural proteins for various pathogens. In general, we use mammalian expression systems to generate our VLPs. However, in our hands, mammalian-cell derived CHIK VLP yields were low. CHIK VLP yield was more robust when using A recombinant baculovirus-insect cell system. In general, baculovirus-insect cell systems yield higher amounts of recombinant proteins, which may result from the higher cell densities t...

Virus-like particles (VLPs) are an approved technology for human vaccination. In fact, some of the more contemporarily licensed vaccines, including the human papillomavirus (HPV) and hepatitis&#160;&#914; (Hep&#914;) vaccines employ this approach. VLPs are formed from structural proteins capable of self-assembly. The assembled particles mimic viral morphologies, but cannot infect or replicate because they lack viral genomes. VLPs can be expressed and purified from a number of prokaryotic and eukaryotic systems. A review of the literature revealed that different expression systems are employed at the following rates: bacteria - 28%, yeast - 20%, plant - 9%, insect - 28%, and mammalian - 15%1. Of note, HPV vaccines based on L1 capsid protein are produced in yeast (Gardasil) or in an insect cell system (Cervarix)2. Hep&#914; vaccines, Recombivax and Engerix-&#914;, are also produced in yeast, and are composed of Hep&#914; surface antigen3,4.
We use mammalian and insect cell expression systems to produce VLPs requiring co-expression of multiple structural proteins for assembly. Our work focuses on designing, producing, and purifying VLP-based vaccines against human pathogens: influenza virus, respiratory syncytial virus (RSV), dengue virus (DENV), and chikungunya virus (CHIKV). Our methods are flexible enough to allow for co-expression of the multiple structural proteins from multiple expression plasmids, or a single expression plasmid (Figure 1). Previously, we produced and purified H5N1 VLPs assembled from the co-expression of plasmids encoding influenza hemagglutinin (HA), neuraminidase (NA), and matrix (M1) in human embryonic kidney 293T cells5,6. The genes were codon-optimized for expression in mammalian cells and cloned into pTR600, a eukaryotic expression vector containing the cytomegalovirus immediate-early promoter plus intron A for initiating transcription of eukaryotic inserts and the bovine growth hormone polyadenylation signal for termination of transcription7. A similar approach using the three-plasmid co-expression of HA, NA (Figure 1A) and an alternative viral matrix protein, HIV Gag p55, was used for generation of human seasonal influenza subtype H3N2 VLPs in this study and has been shown to generate VLPs of similar size as influenza particles8. Although influenza vaccines predominantly elicit anti-HA antibodies, the addition of influenza neuraminidase mediates sialidase activity to enable VLP budding from transfected cells9 and also present additional immunogenic targets. To produce RSV VLPs, we also selected the unrelated core of HIV Gag to design prototypical vaccines that present exclusively RSV surface glycoproteins to further demonstrate flexibility of VLP formation using HIV Gag, as previously described and characterized by electron microscopy6,10. Others have previously shown that VLPs presenting RSV glycoproteins can be assembled using various viral components from Newcastle Disease virus (NDV)11, and influenza matrix12. Full-length surface glycoprotein sequences were utilized in this study to retain native conformations that may be necessary for functional receptor binding and antibody recognition assay through enzyme-linked immunosorbent assay (ELISA).
Our examples for use of single plasmid expression systems to generate particles are DENV and CHIKV. In the case of DENV, we can produce subviral particles (SVPs) with no capsid in 293T cells from a single plasmid containing a prM/E structural gene expression cassette13. The term SVP is used to denote the lack of a core or capsid protein in the assembly of viral structural proteins. CHIK VLPs can also be produced using a single plasmid containing a CHIKV structural gene cassette, encoding capsid and envelope proteins, or in insect cells by infecting with a recombinant baculovirus encoding the same structural gene cassette optimized for insect cell expression (Figure 1B-C).
The end result of the expression approaches discussed above is the release of VLPs into cell culture medium that can then be purified via ultracentrifugation through a 20% glycerol cushion. In this report, we present methods to express and purify these VLPs from mammalian and insect cell systems.

<table><tbody><tr><td>Plasmid Maxi Kit</td><td>Qiagen</td><td>12163</td><td></td></tr><tr><td>DMEM</td><td>Cellgro</td><td>10-013</td><td></td></tr><tr><td>Lipofectamine</td><td>Life Technologies</td><td>L3000075</td><td>Lipofectamine 2000, Lipofectamine 3000</td></tr><tr><td>Opti-MEM I</td><td>Life Technologies</td><td>31985062</td><td></td></tr><tr><td>Bac-to-Bac Baculovirus Expression System</td><td>Life Technologies</td><td>10359-016</td><td></td></tr><tr><td>Cimarec I 6 multipoint stirrer plate</td><td>ThermoFisher Scientific</td><td>50094596</td><td></td></tr><tr><td>Polyclear ultracentrifuge tubes</td><td>Seton</td><td>7025</td><td>Confirm appropriate tubes for ultracentrifuge and bucket size</td></tr><tr><td>Micro BCA Protein Assay Kit</td><td>ThermoFisher Scientific</td><td>23235</td><td></td></tr><tr><td>Phosphate citrate buffer</td><td>Sigma</td><td>P4922</td><td></td></tr><tr><td>o-phenylenediamine dihydrochloride&amp;nbsp;</td><td>Sigma</td><td>P8287</td><td></td></tr><tr><td>0.22 &amp;mu;m vacuum filter top (500 ml)</td><td>Nalgene</td><td>569-0020</td><td></td></tr><tr><td>Glycerol</td><td>Sigma</td><td>G5516</td><td></td></tr><tr><td>H&lt;sub&gt;2&lt;/sub&gt;SO&lt;sub&gt;4&lt;/sub&gt;</td><td>Sigma</td><td>339741&amp;nbsp;</td><td></td></tr><tr><td>Sf-900 II SFM</td><td>ThermoFisher Scientific</td><td>10902-096</td><td></td></tr></tbody></table>

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.

VLP yields were variable by viral antigen construct design. In this protocol, we have demonstrated use of insect and mammalian cells for production of SVP or VLPs in supernatant and purification by ultracentrifugation. Four subtypes of DENV prM/E structural gene expression cassettes were used to construct the versions of DENV SVPs (demarcated as 1-4) in Table 1 and demonstrate a range between 1.1-2.6 mg of total protein in 0.6 ml volumes. For VLPs that require a three gen...

Watch this Scientific Journal Video about Expression and Purification of Virus-like Particles for Vaccination at JoVE.com

Expression and Purification of Virus-like Particles for Vaccination

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 ...

expression-and-purification-of-virus-like-particles-for-vaccination

Universidad de Cartagena UDEC

Research

JoVE Journal

Biology

21.8K Views.  University of Georgia. 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.

Video: Expression and Purification of Virus-like Particles for Vaccination

Paramyxoviruses for Tumor-targeted Immunomodulation: Design and Evaluation Ex Vivo

Successful cancer immunotherapy has the potential to achieve long-term tumor control. Despite recent clinical successes, there remains an urgent need for safe and effective therapies tailored to individual tumor immune profiles. Oncolytic viruses enable the induction of anti-tumor immune responses as well as tumor-restricted gene expression. This protocol describes the generation and ex vivo analysis of immunomodulatory oncolytic vectors. Focusing on measles vaccine viruses encoding bispecific T cell engagers as an example, the general methodology can be adapted to other virus species and transgenes. The presented workflow includes the design, cloning, rescue, and propagation of recombinant viruses. Assays to analyze replication kinetics and lytic activity of the vector as well as functionality of the isolated immunomodulator ex vivo are included, thus facilitating the generation of novel agents for further development in preclinical models and ultimately clinical translation.

This protocol describes a detailed workflow for the generation and ex vivo characterization of oncolytic viruses for expression of immunomodulators, using measles viruses encoding bispecific T cell engagers as an example. Application and adaptation to other vector platforms and transgenes will accelerate the development of novel immunovirotherapeutics for clinical translation.

Successful cancer immunotherapy has the potential to achieve long-term tumor control. Despite recent clinical successes, there remains an urgent need for ...

Cancer Research

Production of High-Titer Recombinant Newcastle Disease Virus from Allantoic Fluid

Newcastle disease virus (NDV), also known as avian orthoavulavirus serotype-1, is a negative sense, single-stranded RNA virus that has been developed both as an oncolytic virus and a viral-vectored vaccine. NDV is an attractive therapeutic and prophylactic agent due to its well-established reverse genetics system, potent immunostimulatory properties, and excellent safety profile. When administered as an oncolytic virus or a viral-vectored vaccine, NDV elicits a robust antitumor or antigen-specific immune response, activating both the innate and adaptive arms of the immune system. 
Given these desirable characteristics, NDV has been evaluated in numerous clinical trials and is one of the most well-studied oncolytic viruses. Currently, there are two registered clinical trials involving NDV: one evaluating a recombinant NDV-vectored vaccine for SARS-CoV-2 (NCT04871737), and a second evaluating a recombinant NDV encoding Interleukin-12 in combination with Durvalumab, an antiPD-L1 antibody (NCT04613492). To facilitate further advancement of this highly promising viral vector, simplified methods for generating high-titer, in vivo-grade, recombinant NDV (rNDV) are needed. 
This paper describes a detailed procedure for amplifying rNDV in specified pathogen-free (SPF) embryonated chicken eggs and purifying rNDV from allantoic fluid, with improvements to reduce loss during purification. Also included are descriptions of the recommended quality control assays, which should be performed to confirm lack of contaminants and virus integrity. Overall, this detailed procedure enables the synthesis, purification, and storage of high-titer, in vivo-grade, recombinant, lentogenic, and mesogenic NDV for use in preclinical studies.

Here we provide a detailed procedure for production, purification, and quantification of high-titer recombinant Newcastle disease virus. This protocol consistently yields &#62; 6 &#215; 109 plaque-forming units/mL, providing virus quantities appropriate for in vivo animal studies. Additional quality control assays to ensure safety in vivo are described.

Newcastle disease virus (NDV), also known as avian orthoavulavirus serotype-1, is a negative sense, single-stranded RNA virus that has been developed both ...

Utilizing the Antigen Capsid-Incorporation Strategy for the Development of Adenovirus Serotype 5-Vectored Vaccine Approaches

Adenovirus serotype 5 (Ad5) has been extensively modified with traditional transgene methods for the vaccine development. The reduced efficacies of these traditionally modified Ad5 vectors in clinical trials could be primarily correlated with Ad5 pre-existing immunity (PEI) among the majority of the population. To promote Ad5-vectored vaccine development by solving the concern of Ad5 PEI, the innovative Antigen Capsid-Incorporation strategy has been employed. By merit of this strategy, Ad5-vectored we first constructed the hexon shuttle plasmid HVR1-KWAS-HVR5-His6/pH5S by subcloning the hypervariable region (HVR) 1 of hexon into a previously constructed shuttle plasmid HVR5-His6/pH5S, which had His6 tag incorporated into the HVR5. This HVR1 DNA fragment containing a HIV epitope ELDKWAS was synthesized. HVR1-KWAS-HVR5-His6/pH5S was then linearized and co-transformed with linearized backbone plasmid pAd5/&#8710;H5 (GL) , for homologous recombination. This recombined plasmid pAd5/H5-HVR1-KWAS-HVR5-His6 was transfected into cells to generate the viral vector Ad5/H5-HVR1-KWAS-HVR5-His6. This vector was validated to have qualitative fitness indicated by viral physical titer (VP/ml), infectious titer (IP/ml) and corresponding VP/IP ratio. Both the HIV epitope and His6 tag were surface-exposed on the Ad5 capsid, and retained epitope-specific antigenicity of their own. A neutralization assay indicated the ability of this divalent vector to circumvent neutralization by Ad5-positive sera in vitro. Mice immunization demonstrated the generation of robust humoral immunity specific to the HIV epitope and His6. This proof-of-principle study suggested that the protocol associated with the Antigen Capsid-Incorporation strategy could be feasibly utilized for the generation of Ad5-vectored vaccines by modifying different capsid proteins. This protocol could even be further modified for the generation of rare-serotype adenovirus-vectored vaccines.

Here, we present a protocol to generate a proof-of-principle divalent adenovirus type 5 (Ad5) vector Ad5/H5-HVR1-KWAS-HVR5-His6 by utilizing the Antigen Capsid-Incorporation strategy. This vector was demonstrated to exhibit qualitative fitness, the capability to escape Ad5-positive sera in vitro, and the antigenicity as well as immunogenicity to the incorporated antigens.

Adenovirus serotype 5 (Ad5) has been extensively modified with traditional transgene methods for the vaccine development. The reduced efficacies of these ...

Immunology and Infection

A Simple and Efficient Approach to Construct Mutant Vaccinia Virus Vectors

The CRISPR-associated endonuclease Cas9 can edit particular genomic loci directed by a single guide RNA (gRNA). The CRISPR/Cas9 system has been successfully employed for editing genomes of various organisms. Here we describe a protocol for editing the vaccinia virus (VV) genome in the cytoplasm of VV-infected CV-1 cells using the RNA-guided Cas9. RNA-guided Cas9 induces double-stranded DNA breaks facilitating homologous recombination efficiently and specifically in the targeted site of VV and a transgene can be incorporated into these sites by homologous recombination. By using a site-specific homologous vector with transgene(s), a N1L gene-deleted VV with the red fluorescence protein (RFP) gene incorporated in this region was generated with a successful recombination efficiency 10 times greater than that obtained from the conventional homologous recombination method. This protocol demonstrates successful use of RNA-guided Cas9 system to generate mutant VVs with enhanced efficiency.

Vaccinia virus (VV) has been widely used in biomedical research and the improvement of human health. This article describes a simple, highly efficient method to edit the VV genome using a CRISPR-Cas9 system.

The CRISPR-associated endonuclease Cas9 can edit particular genomic loci directed by a single guide RNA (gRNA). The CRISPR/Cas9 system has been ...

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.

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.

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

Bioengineering

Production of High-Titer Infectious Influenza Pseudotyped Particles with Envelope Glycoproteins from Highly Pathogenic H5N1 and Avian H7N9 Viruses

The occasional direct transmission of the highly pathogenic avian influenza A virus H5N1 (HPAI H5N1) and H7N9 to humans and their lethality are serious public health issues and suggest the possibility of an epidemic. However, our molecular understanding of the virus is rudimentary, and it is necessary to study the biological properties of its envelope proteins as therapeutic targets and to develop strategies to control infection. We developed a solid viral pseudotyped particle (pp) platform to study avian influenza virus, including the functional analysis of its hemagglutinin (HA) and neuraminidase (NA) envelope glycoproteins, the reassortment characteristics of the HAs and NAs, receptors, tropisms, neutralizing antibodies, diagnosis, infectivity, for the purposes of drug development and vaccine design. Here, we describe an experimental procedure to establish pps with the envelope glycoproteins (HA, NA) from two influenza A strains (HAPI H5N1 and 2013 avian H7N9). Their generation is based on the capacity of some viruses, such as murine leukemia virus (MLV), to incorporate envelope glycoproteins into a pp. In addition, we also detail how these pps are quantified with RT-qPCR, and the infectivity detection of native and mismatched virus pps depending on the origin of the HAs and NAs. This system is highly flexible and adaptable and can be used to establish viral pps with envelope glycoproteins that can be incorporated in any other type of enveloped virus. Thus, this viral particle platform can be used to study wild viruses in many research investigations.

This protocol describes an experimental process to produce high-titer infectious viral pseudotyped particles (pp) with envelope glycoproteins from two influenza A strains and how to determine their infectivity. This protocol is highly adaptable to develop pps of any other type of enveloped viruses with different envelope glycoproteins.

The occasional direct transmission of the highly pathogenic avian influenza A virus H5N1 (HPAI H5N1) and H7N9 to humans and their lethality are serious ...

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 ...

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 ...

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

A Technique for the Generation of Influenza Virus-Like Particles via a Mammalian System

Source: Arevalo, M. T., et al.&#160; <a href="https://appjove.unicartagenaproxy.elogim.com/v/54041">Expression and Purification of Virus-like Particles for Vaccination.</a> J. Vis. Exp. (2016).
This video demonstrates the generation of influenza virus-like particles (VLPs) in a mammalian system. Upon expressing three eukaryotic expression vectors &#8212; encoding different viral structural proteins &#8212; in suitable mammalian host cells, the expressed viral proteins self-assemble into VLPs on the host plasma membrane and release via budding from the cell surface.

Source: Arevalo, M. T., et al.&#160; Expression and Purification of Virus-like Particles for Vaccination. J. Vis. Exp. (2016).
This video demonstrates the ...

Expression and Purification of Virus-like Particles for Vaccination

Summary

Explore More Videos

Chapters in this video

Paramyxoviruses for Tumor-targeted Immunomodulation: Design and Evaluation Ex Vivo

Production of High-Titer Recombinant Newcastle Disease Virus from Allantoic Fluid

Utilizing the Antigen Capsid-Incorporation Strategy for the Development of Adenovirus Serotype 5-Vectored Vaccine Approaches

A Simple and Efficient Approach to Construct Mutant Vaccinia Virus Vectors

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

Production of High-Titer Infectious Influenza Pseudotyped Particles with Envelope Glycoproteins from Highly Pathogenic H5N1 and Avian H7N9 Viruses

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

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

Generating a Multivalent-Displaying Outer Membrane Vesicle Vaccine

A Technique for the Generation of Influenza Virus-Like Particles via a Mammalian System