virus-like particles as vaccine adjuvants

MOLECULAR BIOTECHNOLOGY Volume 19, 2001

Virus-like Particles as Vaccine Adjuvants 169

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Molecular Biotechnology 2001 Humana Press Inc. All rights of any nature whatsoever reserved. 1073–6085/2001/19:2/169–177/$12.25

*Author to whom all correspondence and reprint requests should be addressed: Wellcome Trust Centre for Human Genetics, Roosevelt Drive,Headington, Oxford OX3 7BN UK. E-mail: [email protected]

PROTOCOL

AbstractVirus-like particles (VLPs) consist of one or more viral coat proteins that assemble into particles. They

can be taken up by antigen presenting cells (APC), peptides derived from them are presented on MHC classI molecules at the cell surface, and thereby prime a CD8+ T cell response, either against the particle-formingprotein itself (such as Hepatitis B surface antigen) or additional peptide sequences that are produced asfusions with the particle-forming protein. This article describes the preparation of Ty-VLPs in Saccharomy-ces cerevisiae, a system that can easily be handled in the laboratory or scaled up for manufacture, and is safein use.

Index Entries: Virus-like particle; Ty-VLP; recombinant sub-unit vaccine; CTL.

1. IntroductionAdjuvants are available to promote the genera-

tion of antibodies to an antigen following immu-nization. However, many of these adjuvants donot enhance priming of cytotoxic T lymphocytes(CTL). The reason for this lies in the existence oftwo alternative antigen processing pathways, lead-ing to stimulation of CD4+ T cells, and in turn tothe generation of antibodies, or stimulation ofCD8+ CTL. In general, exogenous proteins enterthe antigen-presenting cells (APC) by endocyto-sis. Peptides produced by proteolytic degradationof these proteins bind to MHC Class II moleculesthat travel to the surface of the cell before stimu-lating CD4+ T cells. Peptides derived from cytoplas-mic proteins are translocated into the endoplasmicreticulum and bind to MHC class I molecules.When these reach the surface of the APC, theyprime CD8+ CTL. Thus, to generate a CTL responsefollowing immunization it is necessary to feedpeptides into the correct processing pathway. Thiscan be done by expressing the antigen inside theAPC, using a DNA vaccine or recombinant virus.

DNA vaccines can be produced easily, but theCD8+ T cell response may only be moderate (1),particularly in larger animal models and humans(2). Recombinant viruses are more difficult to pre-pare, and there are safety concerns over the use ofsome viruses as vaccines. However, recombinantVLPs are a safe and highly immunogenic alterna-tive. These small particles, consisting of one ormore viral coat proteins, can act as an adjuvant bycarrying peptide sequences inside the APC andfeeding into the “endogenous” processing path-way (3,4), a phenomemon known as “cross-prim-ing.” No additional adjuvant is needed. If VLPsare administered with alum, CTL priming does nottake place, although the antibody response to theVLPs is enhanced (5,6).

The effect of other adjuvants on CTL induction withTy-VLPs has also been studied (6). Mice immunizedwith recombinant Ty-VLPs mixed with aqueous adju-vants (Detox, γ-inulin, galactosaminylmuramyl dipep-tide, or Chemivax) generated CTL responses, but atlevels lower than in the “no adjuvant” group. Other ad-juvants (alhydrogel, algamulin, or SAF-MF) com-

Virus-like Particles as Vaccine Adjuvants

Sarah C. Gilbert


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pletely abrogated CTL responses, although formu-lation of the Ty-VLPs with a very low level ofalum (sufficient to bind 6% of the TyVLP pro-tein) enhanced the CTL response.

Recombinant VLPs are safe and well tolerated.They do not contain any nucleic acids, and areproduced by the over-expression of one or moreviral proteins in the absence of the rest of the viralgenome. They are therefore noninfective, and areproduced using an expression system (S.cerevisiae) that can easily be handled in the labo-ratory and is completely safe to use.

Many viral coat proteins have been used to pro-duce VLPs. In some cases it is of interest to inducean immune response against epitopes containedwith the particle-forming protein itself. Thewidely used vaccine against hepatitis B consistsof VLPs, and studies have been carried out usingVLPs to immunize against HIV (7), Norwalk vi-rus (8) and rotavirus (9). However, some VLP-forming proteins will tolerate the addition ofpeptide sequences without losing the ability toform particles, and then act as an adjuvant for theadditional sequence. Some, such as the hepatitisB core antigen (10) and the cowpea mosaic virus(11) can only carry a small number of amino ac-ids, but there are several systems for producingVLPs carrying long N- or C-terminal extensions.

The most successful malaria vaccine tested todate, RTS,S is based on the C-terminus of circum-sporozoite surface protein (CSP) from Plasmo-dium falciparum fused to hepatitis B surfaceantigen (HBs), formed into particles with addi-tional copies of the HBs protein, and combinedwith a novel adjuvant system (SBAS2). The vac-cine has been shown to be safe and well tolerated,and induces interferon-gamma-secreting CD4+ Tcells and antibodies (12–15).

A number of chimeric particles have been con-structed using the NS1 protein of bluetongue virusexpressed in recombinant baculovirus (16). Up to116 amino acids were inserted at the C-terminus,resulting in the formation of tubules with the for-eign sequence exposed on the surface. Wheninsect cells were co-infected with three differentrecombinant baculovirus constructs, hybrid tu-bules containing the foreign sequence from all

three were produced, proving this to be a flexiblesystem. Chimeric VLPs have also been producedusing papillomavirus proteins (17). Up to 43 kDacan be fused to the L2 minor capsid protein, whichis then co-expressed with the L1 major capsid pro-tein, resulting in the formation of hybrid particlescarrying the foreign sequence. However, as theVLPs consist mainly of the nonrecombinant L1,the dose of foreign sequence per VLP is reduced.

Ty-VLPs are formed from a single protein spe-cies derived from the p1 protein of the TyAretrotransposon found in S. cerevisiae. The p1protein can be expressed from a multicopy plas-mid in S. cerevisiae, resulting in the formation ofhollow, spherical 30 nm particles in the cyto-plasm of the yeast. VLPs still assemble whenonly the first 381 amino acids of the p1 sequenceare expressed, and foreign sequences of up to 42kDa can be fused to the C-terminus without pre-venting particle formation (18). In one study,strings of epitopes were synthesized, linked to-gether, and fused to TyA p1. The longest epitopestring tested contained 229 amino acids, and VLPyield was not reduced by the presence of the for-eign sequence (19). As Ty-VLPs are produced inS. cerevisiae, it is highly likely that the proteinparticles have mannose residues attached. It isthought that this contributes to the adjuvant ef-fect by enhancing uptake of Ty-VLPs into anti-gen-presenting cells, although the mechanism ofup-take has not been studied in detail.

In a study designed to compare CTL inductionusing 10 different vaccine/adjuvant combinations,only lipopetides and Ty-VLPs carrying a foreignepitope consistently primed CTL responses in allimmunized animals (20). After a single subcuta-neous dose of 100 µg Ty-VLPs carrying a CTLepitope, net specific lysis ranged from 42% to62% when assayed at an effector:target (E:T) ratioof 40:1. Reducing the dose to 20 µg resulted in netspecific lysis levels of 25% to 42% at the sameE:T ratio. Mucosal immunization of macaqueswith a Ty-VLP carrying SIV p27 resulted in in-duction of MHC Class I restricted CD8+ CTL re-sponses (21). In this study short-term cell lineswere grown from peripheral blood mononuclearcells (PBMCs) following repeated immunization



with Ty-VLPs carrying the SIV p27 sequence. Be-tween 33 % and 42% of cell lines were positivefor SIV p27-specific lysis, whereas no positivelines were present in animals that had been immu-nized with Ty-VLPs lacking the SIV p27 se-quence. High titer neutralizing antibodies to HIVV3 were produced when recombinant Ty-VLPswere injected with alum (22). Clinical trials havebeen carried out with Ty-VLPs carrying HIV se-quences, with and without alum. The vaccine wassafe and well tolerated. Antibodies andlymphoproliferative responses were generatedwhen alum was used (23,24), and CTL wereprimed when alum was omitted (25).

It may sometimes be desirable to induce eitherT cell responses or antibodies against the sameprotein sequence. This can be done with recombi-nant Ty-VLPs by immunizing with or withoutalum. However, if only an antibody response isrequired, this is probably best achieved by a sim-pler approach such as immunizing with proteinmixed with an adjuvant.

This article describes the production of recom-binant Ty-VLPs. Some sequences have beenfound to prevent particle formation or reduce theexpression of the recombinant protein to a levelthat makes purification difficult. However, themajority of sequences that have been testedresulted in particle formation. In order to produceTy-VLPs, DNA encoding the desired sequenceis fused in frame to the C-terminus of the trun-cated TyA p1 gene, which is controlled by a ga-lactose inducible promoter. S. cerevisiae is thentransformed with the new construct, togetherwith a second plasmid that improves VLP yieldafter galactose induction. The transformed yeastis then grown to high cell density and Ty-VLPproduction is induced by the addition of galac-tose. The yeast cells are harvested and lysed torelease the Ty-VLPs, which are purified by den-sity gradient centrifugation using sucrose. Theprotocol, which can easily be carried out in alaboratory, produces Ty-VLPs of a quantity andpurity suitable for animal immunization or invitro studies. If material for clinical trials is re-quired, the production process can be scaled upand carried out in a GMP facility.

2. Materials

2.1. Plasmids and Yeast Strain

Plasmid pOGS40 contains a C-terminal trun-cated p1 gene (amino acids 1–381) in a multicopyyeast plasmid (Fig. 1). A BamHI site immediately5' to the stop codon allows an additional codingsequence to be inserted. Plasmids pOGS 40, 41,and 42 have the BamHI site in reading frames 1,2, and 3, respectively, but are otherwise identical.These three plasmids contain the yeast LEU2marker that will transform a leu2 auxotroph to leu-cine independence.

The p1 gene is under the control of the strong,galactose-inducible PAL promoter. In theabsence of galactose in the yeast cell, the GAL80protein binds to the GAL4 protein. When galac-tose is present, GAL4 is released and binds to anupstream activation sequence in the PAL pro-moter, thereby activating transcription. How-ever, the amount of GAL4 protein present in theyeast cell is low, and limits the induction oftranscription from a multicopy plasmid contain-ing a galactose-inducible promoter. PlasmidpUG41S contains the GAL4 gene under thecontrol of a galactose-inducible promoter andthe URA3 marker. When a yeast cell is trans-formed with this plasmid as well as pOGS40,the amount of GAL4 protein available to acti-vate transcription after galactose induction isincreased, resulting in the production of largeramounts of the p1 fusion protein, which then as-sembles into Ty-VLPs.

The yeast strain for Ty-VLP production mustbe auxotrophic for leucine and uracil (leu2,ura3). It is also advantageous if it has mutationsin intracellular proteases, which could other-wise degrade the Ty-VLPs during the purifica-tion process. Yeast strain MC2 is a suitablestrain, but is also auxotrophic for tryptophan.Therefore, after transformation with pUG41Sand a derivative of pOGS40, tryptophan mustbe included in the selective medium.

The plasmids described above and the yeaststrain MC2 may be obtained from Dr. Guy Layton,British Biotechnology Ltd., Watlington Road,Cowley, Oxford OX4 5LY, UK.


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2.2. Yeast Transformation1. YEPD medium: Yeast extract 10 g/L, peptone

20 g/L, glucose 20 g/L, autoclaved.2. Sterile distilled water.3. 40% polyethylene glycol 3000 solution, autoclaved.4. Selection plates: 6.7 g/L yeast nitrogen base

(YNB), 10 g/L glucose, 20g/L agar, 1 M sorbi-tol. Autoclave and cool to 50°C. Filter sterilizea 1% solution of tryptophan and add 2 mL/L.Swirl to mix and pour plates.

5. 20% sucrose, autoclaved.6. Biorad Gene Pulser electroporation apparatus

and 0.2 cm gap cuvets.

2.3. Growth of Yeastto Induce Ty-VLP Production

1. Growth medium: 6.7 g/L YNB, 10 g/L glucose,autoclaved. Add 2 mL/L 1% tryptophan, filtersterilized, just before use.

2. Induction medium: 6.7 g/L YNB, 3 g/L glu-cose, 10 g/L galactose, autoclaved, with tryp-tophan added just before use as above.

3. TEN buffer: 100 mM Tris pH7.4, 10 mM EDTA,1.4 M NaCl. Autoclave and chill before use.

2.4. Yeast Cell Disruption1. Glass beads, 40 mesh (BDH), autoclaved.

2.5. Purification of Ty-VLP1. 60% sucrose in TEN buffer, autoclaved.2. 35% sucrose in TEN buffer, autoclaved.3. Prepared dialysis tubing.

2.6. Assessment of Ty-VLP Yield and Purity1. SDS PAGE minigel apparatus and reagents.2. Denaturing sample buffer.3. Coomassie stain and destain.

3. Methods3.1. Preparation of Plasmids

1. To make Ty-VLPs expressing additional aminoacid sequences, the required sequence is addedat the BamHI site of pOGS 40, 41, or 42. Thissequence may consist of single B or T cellepitopes, strings of epitopes joined together, orwhole antigens. If defined epitopes are to beused, the DNA sequence coding for the epitopeshould be synthesized as oligonucleotides.These should be designed to fuse in frame toone of the pOGS vectors (see Fig. 1). No stopcodon is necessary as this is present after theBamHI site in the vectors. If the epitope isderived from an organism with a codon usagevery different from that of yeast, the insertshould be designed using yeast codon bias.

2. It is then necessary to check for the possibilityof the formation of secondary structures withinthe oligo’s, and alter the sequence if necessary(see Note 1). It is preferable to synthesizeoligo’s for both the top and bottom strand ofthe insert. They should be purified after syn-thesis (HPLC or gel purification) and 5' phos-phate groups should be added. The oligo’s arethen annealed by heating 5 pmol of each in20 µL water to 95°C, and allowing them to coolslowly. Five microliters of this mixture is thenligated to 0.1 µg of the appropriate pOGS vec-tor, which has been digested with BamHI and

Fig. 1. (A) Plasmid map of pOGS40. (B) the DNAsequence around the BamHI site at the 3'end of theTyAp1 gene, and an example of a pair of oligonucle-otides designed to ligate into the BamHI site, encod-ing the HLA-B35 CTL epitope KPNDKSLY from theliver stage antigen 1 gene of Plasmodium falciparum.



phosphatased to prevent religation of the vec-tor. Competent cells of Escherichia coli arethen transformed with the ligation mix, andplasmids prepared by standard methods.

3. For very small inserts it is not possible to deter-mine the orientation by restriction digests, sothe plasmid must be sequenced (see Note 2).All oligo inserts need to be sequenced to con-firm that the newly synthesized sequence doesnot contain errors.

4. The maximum length for oligo synthesis isaround 80 bases if a reasonable yield is to beobtained. For an insert of greater than 30 aminoacids, design two oligo’s for the top strand andtwo for the bottom, with an overlap of eightbase pairs in the center. If epitope strings ofmore than 60 amino acids are required, it ispreferable to construct a series of cassettes con-taining three or four epitopes in each, with aBglII site at the 5' end and a BamHI site at the 3'end. Each cassette can be assembled in a clon-ing vector and sequenced. The first cassette isthen introduced at the BamHI site of pOGS40,leaving a single BamHI site at the 3' end for theaddition of further cassettes.

3.2. Yeast TransformationThere are several methods by which yeast cells

may be transformed. The method given below,electroporation, needs little technical expertise butrequires the use of an electroporation apparatus.Other methods for yeast transformation (lithiumacetate, sphaeroplasting) are given in (26).

1. Grow yeast MC2 in YEPD (50 mL in a 250 mLconical flask). Monitor cell density by remov-ing a sample aseptically, diluting the sample 1in 10 with water and measuring OD600 in a spec-trophotometer, using water as a blank. The cellsare ready to use when the OD600 of the dilutedsample is between 0.4 and 0.6 (see Note 3).

2. Harvest by centrifugation in sterile 50 mL Fal-con tubes in a benchtop centrifuge, 10 min at1900g, 4°C.

3. Resuspend the pellet in 50 ml ice-cold steriledistilled water, pellet cells again. Repeat thiswash step twice more.

4. Resuspend the cells in 5 mL ice-cold sterile dis-tilled water. Chill on ice.

5. Add the two plasmids for cotransfection(pOGS40 derivative and pUG41S) to an elec-troporation cuvet. Use 5 µg of each plasmid ina total volume of 10 µL sterile distilled water.Chill on ice.

6. Add 100 µL yeast cells and 50 µL 40% PEG3000 solution to the cuvet containing the DNA.Mix, incubate on ice for 10 min.

7. Set the following values on the gene pulserapparatus: resistance 1000, capacitance 25,voltage 0.8. Dry the cuvet and pulse.

8. Add 0.5 mL 20% sucrose to the cuvet, mix andtransfer the contents to a sterile Eppendorf tube.

9. Plate out all the yeast cells on selection plates,spreading approx 200 µL per plate. Allow thesurface of the plates to dry, then incubate at30°C. Colonies should appear within 1 wk (seeNote 4).

10. When transformants appear, streak each one ona fresh selection plate (sorbitol need not beincluded at this stage) and incubate for 2–3 d.For short-term storage these plates can besealed with Parafilm and held at 4°C. Forlonger-term storage of transformants prepareglycerol stocks (see Note 5).

3.3. Growth of Yeastto Induce Ty-VLP Production

1. Inoculate 50 mL of growth medium in a 250 mLconical flask with a loopful of cells grown froma single transformant, or a glycerol stock.

2. Incubate at 30°C in a shaking incubator for atleast 24 h.

3. Remove a sample of the culture aseptically.Dilute 100 µL of the cells with 900 µL waterand measure the OD600 in a spectrophotometer,using water as a blank. The OD600 of the dilutedsample should be at least 0.2. If it is below this,return the flask to the incubator and continuemonitoring (see Note 6).

4. Inoculate 500 mL of induction medium in a 2 Lconical flask with the 50 mL culture. Incubateat 30°C with shaking for 18 h. Monitor OD600.If it is less than 0.2, continue to incubate theculture for a further 6 h.

5. Harvest the induced culture by centrifugationat 4°C, for example 15 min at 5000g in aBeckman J-6MC centrifuge with JS4.2 rotor.Discard the supernatant and resuspend the cells


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in 50 mL chilled TEN buffer. Transfer to a 50mL Falcon tube and pellet the cells in abenchtop centrifuge, 5 min at 1900g, 4°C.

6. Repeat the wash step once more, and discardthe supernatant. The cells can be frozen at –70°Cat this point for assay at a later date if required.

3.4. Yeast Cell Disruption1. Resuspend the cell pellet in 2 mL ice-cold TEN

buffer and add 6 g sterile glass beads.2. Vortex vigorously for 5 min (see Note 7).3. Centrifuge the homogenate for 5 min at 1900g,

4°C in a benchtop centrifuge. Remove thesupernatant to a 50 mL Falcon tube, on ice.

4. Add another 2 mL of TEN buffer to the pelletand vortex vigorously for 5 min. Centrifuge andadd the supernatant to that collected in step 3.

5. Add 1 mL of TEN buffer to the pellet, vortex,centrifuge, and add the supernatant to that col-lected in steps 3 and 4.

6. Centrifuge the supernatant for 20 min at13,000g, 4°C, for example, in a Beckman J2-MC centrifuge with JA12 rotor.

7. Carefully transfer the supernatant (clarifiedlysate) to a clean tube and keep on ice. Re-tain a sample of the supernatant for SDSPAGE analysis.

3.5. Purification of Ty-VLPs1. Add 2.75 mL of the clarified lysate to a 3.2 mL

polycarbonate ultracentrifuge tube. Set a Gil-son P200 pipet to 250 l, and carefully underlay250 l of 60% sucrose in TEN buffer at the bot-tom of the tube (see Note 8).

2. Prepare a balance tube and centrifuge at 417,000gfor 17 min at 4°C, in a Beckman Optima TLbenchtop ultracentrifuge with TLA 100.4 rotor.Acceleration and deceleration rates should beset to mid range (5).

3. The Ty-VLPs will accumulate just above the60% sucrose layer, and it may be possible tosee a milky band in the tube at this point.Remove the supernatant above this, and discard.If no milky band is visible, it will still be pos-sible to distinguish the interface between thesucrose and the supernatant. Remove the super-natant leaving 2–3 mm above the interface.

4. Resuspend the material remaining in the tubein 1 mL ice-cold TEN buffer.

5. Add 1.5 mL of 35% sucrose in TEN to a cleancentrifuge tube. Underlay with 250 µL 60%sucrose in TEN as before. Slowly layer theresuspended material from step 4 on top of the35% sucrose.

6. Prepare a balance tube and centrifuge at 417,000gfor 40 min at 4°C.

7. Repeat step 3. Resuspend the remaining mate-rial in 1 mL ice-cold TEN buffer. Retain a sam-ple for SDS PAGE analysis.

8. Remove the sucrose from the Ty-VLPs byovernight dialysis in TEN buffer, at 4°C. Meas-ure the protein concentration using, for exam-ple the Biorad Protein assay Kit. Aliquot andfreeze samples for storage at –70°C (see Note 9).

3.6. Assessment of Ty-VLP Yield and Purity1. Prepare a 10% SDS PAGE minigel.2. Prepare the samples for analysis in denaturing

loading buffer. Boil the samples for 2 min. Thiscauses the Ty-VLPs to dissociate into proteinmonomers which can easily be visualized afterSDS PAGE. Samples should include the clarifiedlysate (diluted 1 in 10) and the final preparation,with size standards covering the range 6–175 kDa.It is also helpful to include Ty-VLPs preparedfrom yeast cotransformed with the pOGS40 vec-tor and pUG41S, as a positive control for galac-tose induction and VLP preparation.

3. Run the SDS PAGE gel, loading 2, 5, and 10 µLof each sample. Stain the gel with Coomassie.

4. Ty-VLP momomers should be clearly visibleon the destained gel, although the yieldobtained with fusions to the p1 sequence maybe less than that obtained using the pOGS40vector alone. TyA from pOGS40 has an appar-ent molecular weight of around 65 kDa (Fig. 2).If no Ty-VLPs are present, but a band of theexpected size is found in the clarified lysate,the fusion of the additional sequence has inter-fered with particle formation.

3.7. Scale Up of PurificationThe purification method described above yields

around 1 mg of purified Ty-VLPs per 500 mL cul-ture, if the additional sequence does not result in adecreased expression level. As only 50–100 µg isrequired to immunize a mouse, and the methodcan easily be carried out with four 500 mL cul-



tures at the same time, this will provide sufficientTy-VLPs for many experiments.

If larger amounts are required, the same proce-dure can be scaled up by using a full-size ultra-centrifuge for the density gradient centrifugation.Purity can be improved by size exclusion chroma-tography after density gradient centrifugation,and, if necessary, the preparation can be concen-trated by ultrafiltration (18). This method can beused to produce Ty-VLPs suitable for immuniza-tion of humans.

3.8. Assessment of Immunogenicityin Mice

Ty-VLPs injected without adjuvant prime a cy-totoxic T cell response to the epitopes carried on

the particles. They may be administered via thesubcutaneous, intramuscular, intranasal, intrave-nous, or intraperitoneal routes at a dose of 20–100 µg (27). Two weeks after immunization,splenocytes are prepared and tested in a chromiumrelease lysis assay or interferon γ ELISPOT assay(1). Ty-VLPs can be used to load target cells for achromium release assay (20). An example of theresults of a chromium release lysis assay is shownin Fig. 3.

Subcutaneous injection of Ty-VLPs with alumprevents the induction of a CTL response, butenhances antibody production (6).

4. Notes1. If the oligo that has been designed contains

sequences that can pair internally to form largesecondary structures (hairpins) it is not likelythat the insert will anneal as desired when theoligo’s are ligated into the vector. There aremany computer packages that can check this,such as those designed to look at RNA second-ary structures, or PCR primer design packages.If a strong secondary structure could be formed,it is necessary to change bases within thesequence to prevent secondary structure forma-tion without changing the amino acid sequence.

2. Suitable primers to sequence inserts in thepOGS plasmids are GAA GAA TGA TTCTCG CAG CTA (anneals to 3' end of p1 pro-tein) and AGA AAA AAA TTG ATC TATCG (anneals to the 5' end of the transcrip-tion terminator).

3. Grow a 10 mL culture inoculated with a loopfulof yeast cells taken from a YEPD plate inYEPD overnight. The next morning set up aseries of 50 mL flasks of YEPD, inoculatingwith a range of volumes (0.05–1.0 mL) fromthe overnight culture. Incubate all the flasksand monitor OD600 after 3–6 h, by which timeone of the flasks should contain cells at an appro-priate density to prepare for electroporation.

4. Cotransformation with two plasmids is a veryinefficient process and few transformants areexpected to grow. The DNA used for transfor-mation should be highly pure, preferably pre-pared using Qiagen columns. Carrying outseveral replicates with each pair of plasmidscan increase chances of success.

Fig. 2. Coomassie stained SDS PAGE gel showingpurified and unpurified Ty-VLPs. Lanes 1 and 7: mo-lecular weight markers A, 175 kD; B, 83 kD; C, 62kD; D, 47.5 kD; E, 32.5 kD. Lanes 2–4: total yeastsoluble protein after galactose induction. Lane 2,untransformed yeast; lane 3, yeast transformed withpOGS40 and pUG41S; lane 4, pOGS40 with 229amino acid C-terminal fusion and pUG41S. Lanes 5and 6: Ty-VLP monomers after sucrose gradient puri-fication. Lane 5, pOGS40; lane 6, pOGS40 with 229amino acid C-terminal fusion.


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5. Grow each transformant in 50 mL growthmedium, to late log or early stationary phase(OD600 of 1 in 10 dilution between 0.2 and0.6). Centrifuge and resuspend in 5 mL growthmedium containing 15% glycerol. Aliquot 1 mLper vial and freeze on dry ice. Store at –70°C.To use, thaw in warm water and transfer togrowth medium.

6. Some cultures are slow to grow, and the initialgrowth phase can take up to 3 d.

7. It is essential to break all the yeast cells open.Press the tube hard onto the vortex mixer, hold-ing the tube at an angle. Check for cell break-age by examining the cells under a phasecontrast microscope. Intact cells appear bright,broken cells are dark.

8. The 60% sucrose solution is very viscous, sofill the pipet tip and dispense the solution veryslowly. The pipet tip should be placed at thebottom of the centrifuge tube before starting todispense the sucrose.

9. Ty-VLPs should not be repeatedly frozen andthawed, as this disrupts the particle structureresulting in loss of immunogenicity. It is there-fore most convenient to divide the sample into100 µg aliquots before freezing.

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Fig. 3. CTL response in mice. Balb/c mice wereimmunized with a single subcutaneous injection of 100µg Ty-VLPs carrying a single CTL epitope from theCS gene of Plasmodium berghei (SYIPSAEKI).Spleens were taken at wk 2, 4, or 10 after immuniza-tion. No adjuvant was used, and the assay was carriedout after in vitro restimulation for 1 wk with the ap-propriate peptide, using peptide loaded target cells atan effector: target ratio of 40:1.



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virus-like particles as vaccine adjuvants

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