key: cord-0007979-xwbcfh6p
authors: Roy, Polly; Noad, Rob
title: Virus-Like Particles as a Vaccine Delivery System: Myths and Facts
date: 2009-12-30
journal: Pharmaceutical Biotechnology
DOI: 10.1007/978-1-4419-1132-2_11
sha: efb26217d5a2446e9b9853144d3c65fb40020cc8
doc_id: 7979
cord_uid: xwbcfh6p

Vaccines against viral disease have traditionally relied on attenuated virus strains or inactivation of infectious virus. Subunit vaccines based on viral proteins expressed in heterologous systems have been effective for some pathogens, but have often suffered from poor immunogenicity due to incorrect protein folding or modification. In this chapter we focus on a specific class of viral subunit vaccine that mimics the overall structure of virus particles and thus preserves the native antigenic conformation of the immunogenic proteins. These virus-like particles (VLPs) have been produced for a wide range of taxonomically and structurally distinct viruses, and have unique advantages in terms of safety and immunogenicity over previous approaches. With new VLP vaccines for papillomavirus beginning to reach the market place we argue that this technology has now ‘come-of-age’ and must be considered a viable vaccine strategy.

There are many infectiousviruses that remain major threats to public health (seeTable 1).Where an effective vaccine exists,vaccination is usually the most cost-effective long-term protection against diseaseand spread for most viruses.The principle ofvaccination is to generate sufficient immunity to protect from infectious disease. Thus the vaccine stimulates the body's natural defensesagainst disease through use of a benign 'decoy' that mimics the virulent pathogen. The more similar a vaccine is to the natural disease, the better the immune response to the pathogen on subsequent exposure. In general, resistance to virus infection depends on the development ofan immune response to antigens present on the surface ofvirions or virus-infected cells. Therefore identification ofprotective antigens is the first step in the development ofeffectiveviral vaccines.

Currently many successfulviral vaccines have been developed and are in use. These vaccines are predominantly based on live attenuated or inactivated viruses. The live attenuated vaccines such as measles, mumps, rubella, oral polio, smallpox, varicella and yellow fever are a weakened form ofthe "wild" viruses. These attenuated virus vaccines rely on limited replication ofthe virus in the host following vaccination. Immune responses induced are similar to those from natural infections and often these vaccines are effective after a single dose. However, such vaccines may cause severe reactions in some patients, which are ofien the result ofthe limited replication ofthe att enuated virus following vaccination. In contrast to attenuated live virus vaccines, inactivated (or killed) vaccines can not replicate, as their genetic material or overall structure are purposefully destroyed. These vaccines are safer than live vaccines but generally not as effective, Thesevaccines are madeaswhole cellvaccines (suchasInfluenza,polio,rabiesand hepatitisA) or asfractionalor subunit vaccines such ashepatitisB. Subunit vaccines are basedon the delivery of only a limited number of viral proteins,often the major protein in the capsidor envelope that is sufficient to conferprotectiveimmunity. Thesevaccines arean incrementalstep saferthan inactivated vaccines becausesubunit vaccines can be prepared independent to the culture of replicating virus. Indeed, any remaining possibilityof incomplete inactivation or batch to batch variation in the safetyof the vaccineis eliminated. However, subunit vaccines havetraditionally suffered from one important drawback; often singleproteins when expressed and purified in the absence ofother viral componentsare less immunogenicthan those that areincorporated into infectious virus.Thisisprobablybecause a proportion of this protein ispresentin a misfoldedconformation relative to the nativeprotein. Thus, more doseswith higher amounts of antigen are required to achievethe samelevel ofprotection. A majoradvance in subunit immunogenproduction hasbeen assembly ofproteinsasvirus-like particles (VLPs) usingprotein expression technologyin yeast, insect or mammalian cells. VLPs are a highly effective type of subunit vaccines that mimic the overallstructure of virus particles without any requirement that they contain infectiousgeneticmaterial. Indeed, manyVLPslack the DNA or RNA genome of the virus altogether, but havethe authentic conformation of viral capsidproteins seenwith attenuated virusvaccines, without anyof the risksassociated with virus replication or inactivation.

VLP preparations are all based on the observation that expression of the capsid proteins of manyviruses leadsto the spontaneousassembly ofparticlesthat arestructurallysimilarto authentic virus.':' In practicalterms,the fact that VLPsmimic the structure of virusparticlesusually means that VLPsshouldelicitstronghumoralresponse and that lowerdoses of antigenrelative to subunit vaccines aresufficient to elicitsimilarprotectiveresponse. In addition to their abilityto stimulate B cell mediated immune responses, VLPshavealso been demonstrated to be highly effective at stimulatingCD4 proliferative and =(CTL) responses.l? Thisfeatureof VLPvaccines is likely to be a major contribution to their effectiveness in the field. It is also becomingincreasingly clear that preciseprime-booststrategies canbe important to how effective vaccinationisasa strategyto control disease.Therefore, the addition of VLPto the 'arsenal' of vaccine strategies for anydisease extends the type ofprime-boostregimethat can beemployed.

To date, VLPs have been produced for many differentviruses that infect humans and other animals (seeTable2 and review)," One of the most strikingfeatures of this group is that it is extremely diverse in terms of the structure of the individualviruses. It includesviruses that havea singlecapsidprotein, multiplecapsidproteinsand thosewith and without lipid envelopes. Clearly not allofthe VLPs that are generated to date are appropriate vaccine targets, some VLPs have been generated to facilitate in fundamental understanding of virus assembly process, morphogenesis or architecture ofviruses. However, an important point remains that the structure of the target virion is not limiting to the successofVLP production. Although various expression systems have been employed for VLP production, this chapter will mainly focus on insect cell culture produced VLPs that are being developed as candidate vaccines. The rationale behind this is that among all expression systems, insect cells, together with baculovirus expressing system, appear to be one of the most promising for VLP technology for development ofviral vaccines (Fig. 1) . 

As stated above, a varietyof protein expression systems are available to express recombinant proteins and particles. Howevercertain criteriafor generationofVLPs as prophylacticvaccines, particularly forhumanviralinfection,mustbe considered. In orderforaVLPto bearealisticvaccine candidate, it needs to be produced in a safeexpression systemthat is easyto scaleup to large-scale production.Table2 shows baculovirus expressed/insect cellproducedVLPsthat havebeendemonstrated to be highlyimmunogenicand potential vaccine candidates.Thisinsectcell-based protein production systemhas manyadvantages for VLP production. Firstly, extremely largeamounts of correctly folded recombinant proteins can be produced in high-density cell-culture conditions in eukaryotic cells. Secondly, baculovirus expression systems havebeen developedfor expression of multiple foreign proteins simultaneously from a single recombinant virus facilitating capsid assembly in each infectedcell. Thirdly. asthe insectcellsthat are usedfor vaccine production can be cultured without the need for manunalian cellderivedsupplements. the risksof coculture of opportun istic pathogens is minimized. Fourthly. the baculovirus used for recombinant protein expression has a narrow host range that includesonly a fewspecies ofLepidopteraand therefore represents no threatto vaccinated individuals. Finally the baculovirus system is amenable to scale-up for largescalevaccine production,"

For a number ofnonenveloped virusesviral capsidsare formed by only one or two major proteins and thus are relatively easyto manipulate for generation of VLPsbyheterologousexpression systems. Examplesofthese are the VLPsformed by the expressionofthe major capsidprotein of Papillomaviruses, Parvoviruses, Calciviruses, Circovirses, Polyomaviruses and Hepatitis E virus ( Table 2 ). All of these viruses are nonenveloped and have a single, virallyencoded protein that forms the major structural component of the virion. Papillomavirus VLPs are among the most completelystudied ofthis collection of VLPs and are at the most advancedstagewith respectto production ofa usefulvaccine. VLP ofPapillomavirusesare formed from the over expressionof the major capsidprotein Ll. lO • 12These particlesare highly immunogenic and are ableto stimulate both humoral and cellmediated immune responses.P'" Human Papillomavirus (HPV) isthe leading causeofcervicalcancer.Globally, approximately70% ofall cervicalcancer casesareassociated with two serotypesofHPY, HPV-16 and HPV-lS. VLPsproduced in insect cellshavebeen used successfully for Phase I and II human clinicaltrials in largenumbers and were shown to be highly efficacious. 1S • 19 Moreover, GlaxoSmithKline's cervicalcancervaccinecandidate (Cervarix:"') targeting HPV 161IS iscurrentlyundergoingPhaseIII clinicaltrialsinvolvingmore than 30,000women worldwide. In this Phase III randomized, double-blinded trial conducted in multiple centres in Denmark, Estonia. Finland, Greece. the Netherlands and the Russian Federation. All vaccinees receivedthe HPV VLPs(HPV-16I1S AS04) asfollows: 15S 10-14yearsold healthygirls and 45S 15-25 yearsold young women receivedthe candidate VLP vaccineaccording to a 0,1,6 month schedule and anti-HPV antibody titers wereassessed. At month seven 100 per cent seropositivity wasachievedin both groups for HPV 16 and IS although average antibody titers for both HPV typeswereat leasttwo-foldhigher in 10-14 year-oldgirls.Thevaccinewastolerated byallpatients and no vaccinerelated seriousadverseeffectswere detected. Further, the follow-upsmdy clearly demonstrated the sustained efficacy of HPV-16I1S VLPs up to 4.5 years. 19 ,20 In conclusion, the bivalent HPV vaccine is highly immunogenic and safe and induces a high degree ofprotection against HPV-16 and HPV-lS infection and associatedcervicallesions.

Thesestudiesarenot onlyan important demonstrationofthe effectiveness ofHPVVLP vaccine, and that multi-serotype VLPs are effective, but also highlight the fact that insect cell produced VLPsare a realisticalternativeashuman vaccines againstviraldisease. It should alsobe mentioned at this point that a tetravalent (HPV-61III1611S)VLP vaccine, Guardasil'" (Merk),produced in yeastcellswasapproved by FDA in]une 2006 for use in women aged 9-26.

VLP vaccinesfor variousdiseases causedby parvovirusinfections are alsoat an advancedstage although as yet none have undergone such large scaletrials as those reported for HPV. Synthesis ofmajor structural proteins VP2 ofcanine parvovirus (CPV) and porcine parvovirus (PPV) led to assembly ofVLPs in insect cells. 21 . 22 Vaccinationtrials of CPV VLPs in dogs and PPV VLPS in pigswere highly encouraging.i':" In one efficacy assaydogs that receivedaslittle as or 10~or 25~ofCPV VLP werecompletelyprotected from virus infection when challengedwith virulent virus. Furthermorea singlesubcutaneousdose00~sameCPV VLP with 50~ISCOM adjuvant wasableto protect mink againstchallengewith the anti-genically similarvirus,mink enteritisvirus (MEV),2l Similarlyit has been reported recently that a singleimmunization with 0.7~ofPPV (porcine starin) VLPs yielded complete protection in targeted animals against infectious PPV strains.P Indeed microgram doses ofVLPs in gilts were not only highly immunogenic. but were alsoveryefficientin preventing trans-plancentalvirus transmission and significantly reduced the number of reproductive failures. In addition, the feasibilityof safelarge-scale production of the porcine parvovirusVLPvaccine hasbeen establishedcomplyingwith the EuropeanPharmacopoeia requirements,"

Calicivirusstudies have relied heavilyon the production of proteins in heterologous systems mainly due to the fact that it is not yet possible to grow the virus in cell culture. Thus, VLP to Norwalk-like viruses have been extremely useful as sources of diagnostic antigen to monitor diseaseoutbreaks. Norwalk virus VLP have also been shown to be effective at stimulating IgG, IgA and humoral responsesin mice. 24 VLPsfor Hepatitis E havebeen assembled usinga truncated form of the viruscapsidprotein." In immunization studies in mice these VLPswereable to induce systemic and mucosalimmune responses following oral adminisrrarion.P" Furthermore, oral administration of the Hepatitis E VLPs to cynomologousmonkeys induced IgM, IgA and IgG responses and was sufficient to protect against infection and disease on challengewith virus," Thus there is clear potential for the application of theseVLPsas a vaccinefor hepatitis E.

VLPpreparationsto Circoviruses and Polyomavirusareat a less advanced stage.VLP formation hasbeen reported for Circovirusbut asyetno seriousattempt hasbeenmadeat vaccine production. Vaccination of rabbitswith VLPsfor humanJC virusin the presenceof adjuvantallowedproduction ofa hyperimmuneserumthat effectively neutralizedinfectiousviruspreparadons." However, in the absenceofadjuvant there was no response. This pattern of responseis unusualfor VLPsin general,which often stimulatestrong immune responses evenin the absenceofadjuvant.Indeed, VLPs of murine polyomavirus were able to stimulate a strong immune responsein the absence of adjuvant when administered as a single 610 ng dose. 32 Intriguingly, these particles appear to be particularlystablewith no alteration ofparticle morphology or reduction in immunogenicity even after 9 weeksstorageat room ternperature.P

Viral particles that contain multiple interacting capsid proteins present more of a technical challengethan those that are formed by one or two major capsidproteins. Particularly, it is far more difficultifthe assembling proteins of capsidsare encoded by multiple discretemRNAs, but not processedfrom a singlepolyprotein as in the caseof picornaviruses. This is due the fact that for efficientassembly of a VLP the interacting capsidproteins must be expressed in the vicinity to each other, in other words in the samecell Assembly of VLPsbyprocessingof polyproteins have been achievedboth for poliovirus" and for InfectiousBursaldiseasevirus" usingthe baculovirus expressionsystem. More complex assembly of multilayered, multiprotein VLPs have also been efficiently produced for the members of the Reoviridae. Theseviruseshave capsidsmade up of concentric layers of different capsidproteins. Co-expressionin insect cellsof2-4 ofthese capsid proteins, depending on the virus and the particle made, has allowedthe production ofVLP that are empty of the segmented dsRNA viral genome, but are otherwise indistinguishable from authentic viral particles. 4 • 35The first member of the Reouiridae for which VLPs were described is Bluetongue virus (BTV), an insect transmitted animal virus. This remains the systemin this familyfor which the largestvarietyofdifferentVLPsand recombinant singleantigen subunit immunogens made by baculovirus expression systems has been tested. In addition, the requirement for efficientco-expression of viralcapsidprotein in the sameinsect cellin this systemhas resulted in the development ofbaculovirus multigeneexpression vectors.36.37We will focuson this system in somedetail asit highlightsboth the effectiveness ofVLP vaccines and someof the technological advances that havebeen made for the production ofVLP with complexarchitecture.

Bluetongue disease affects mainly sheep and cattle and is classified as an emergingdiseasein Europe." The disease is causedby bluetongue virus,BTY,which has a multi-layered icosahedral structure formed by nonequimolar amounts of sevenviral proteins (VP1-VP7). Three of these structural proteins (VP1,VP4, VP6) are dispensable for the formation of VLPsas they playonly an enzymaticrole in the virus transcription rnachinery.P The remainingfour structural proteins (VP2, VP5, VP3 and VP7) are organisedin two capsids. The inner capsidactsasa scaffold for the assembly of outer capsidthat is responsible for cellentry and hence contains the major candidate for virus neutralisation." Expression of all four major structural proteins of BTV was achieved by construcing a baculovirus that simultaneously expressed all four proteins.YThe advantage of this approach over co-infection with several baculoviruses each expressing a single protein is that equivalent conditions are achievedin all infected cells. Thus assembly ofVLP is more efficientasexpression 120 100

-. · -10 J,g VlP · · ·. · · 5OI'll VLP is controlled at the level of the cell. rather than the level of the culture as is the case with mixed infections. BTV VLPs (Fig. 2) VLPs from two different serotypes were combined to vaccina te the same animal. In these animals VLPs vaccination provided complete protection against the rwo vaccine serorypes and also partial protection fromchallenge with relatednonvaccine serotypes.The protectiveefficacy ofvaccination in thesetrialsextendedovera long(14 month) period.P'Ihis observationraises the possibilitythat a broad spectrumvaccine againstall 24 BTV serotypes is a possibilitybycombiningVLPsfrom a relatively smallnumber of serotypes.

The BTV system also demonstratesthe efficiency ofVLP vaccines relative to immunization with subunit vaccines based on dissociated antigens or unassembled recombinant antigens. In addition the assembled VLPsthe two componentsofthe BTV outer capsid, VP2 and VP5, were alsopreparedand testedin vaccination studies.While 100~VP2,the majorserotypedetermining antigen,wasonlypartiallyprotectiveforashort duration (75days) againstvirulentviruschallenge, 50~ofVP2 combined with 25~VP5 was protective." In contrast, 10~VLPs (containing only 1-2~VP2) affordeda better level of protection for a much longer duration." Thesestudies demonstrate that assembly of antigensinto VLPsresultsin a more effective immunogen than deliveryof separately isolatedproteins.

In addition to BTV, VLP have also been produced for rotavirus, another member of the Reoviridae. Intriguingly, VLPsformedfrom the twoinner structuralproteinsaloneof the rotavirus capsid have been shown to be effective immunogens in animal modelsY-48Indeed in mice even intrarectal immunisationwhich inducesa localmucosalresponse issufficient for protection from rotavitus infection .v The data from these immunogenicityexperiments are encouragingand it is possible rotavirusVLP mayprovidea viablealternative to the livevitus vaccine for rotavitus.

Manypathogenicviruses suchasInfluenza, HIV and HepatitisC aresurroundedbyan envelope, a membranethat consists of a lipid bilayerderivedfrom the host cell,insertedwith vitusglycoprotein spikes. Theseproteins are the targetsofneutralizingantibodiesand areessential components ofvaccine. Due to the inherent properties of lipid envelope, assembly ofVLPs in insect cellsfor thesevitusesis a different type oftechnicalchallenge to those produced for vituseswith multiple capsids. Nevertheless, efficient formation ofVLPs of a number of enveloped viruses in insectcells hasbeen reported.Forexample, VLPsof HepatitisC virus,several retroviruses, SARSCoronavirus and influenza A havedemonstratedcorrectassemblyof the the lipidenvelope with theglycoproteins inserted.so-ss Indeed,for retroviruses, it hasbeenpossible to producehybridVLPsthat contain the gagcapsidprotein fromone virus(SIV) and the envelopeprotein from another (HIV)S6 in insect cells. Although none of the retrovirus derivedVLPsare yet at the stagethat they are beingusedin clinicalvaccinetrials,initial experiments in anitnalmodelsare promising. s7 . s8

VLPs for SARS Coronavitus as a basisfor vaccinationwere produced rapidlyfollowing the SARSoutbreak in 2002-2003. S4.SS Howeverthe control ofSARS Coronavirusbyepidemiological measures, continued lackof re-emergence of the virus,and difficulties workingdirectlywith the virus haveseverely limited the developmentof SARSVLPs as vaccine. Despite this, anti-serum raisedin mice againstinsect cellderivedSARSVLPswereableto neutralizea retroviruspseudotyped with the SARSS protein (Fig.3) .

The Hepatitis C VLPs (Fig. 1 )havebeen tested in miceand baboonsand shown to be effective at stimulatingboth cellularand humoralimmune responses.?·S3oS9 In one experiment, 6-8 weekold female BALBlc micewere immunizedintramuscularly three times,at three week intervalswith 20~insect cellderivedH CV VLP, produced byco-expressing HCV coreE1-E2. Because of the lackofasuitableanimalmodelfor H CV infectionsa recombinantvaccinia vitusexpressing HCV structural proteins (vvHCV.S)wasusedasa modelsystem. Vaccinated micewerechallenged three daysafterthe finalimmunizationwithvvHCV.S and then five dayslaterthe ovaries ofinfectedmice wereharvested and the vaccinia virustitre determined. Fiveout ofseven vaccinatedanimalshad no detectablevacciniavirus in the ovaries at this point. The remainingtwo anitnalshad five logs lowervacciniatitres compared to control mice'? In addition, this study wasable to demonstrate that the VLPsefficacy wasbasedlargely on its stimulation of CD4+ and CD8+ T-cell responses.

A further study in baboonshas demonstrated that the VLPsare welltolerated and can stimulate broad and long-lastingHCV targeted immune responses.P B 600 , 500 Figure 3 . Summary of production and testing of VlPs to SARS coronavirus. A) left, cartoon and right, electron micrograph of VlPs produced by co-expression of E, M and S proteins of SARS coronavirus . These VlPs were used to raise anti-sera in mice and the ability of these anti-sera to protect against infection with a SARS 5 protein pseudotyped lentivirus were assessed. B) le90 neutralising antibody dilution for SARS S pseudotyped lentiv irus, using sera from 3 mice immun ized with SARS VlP, rotavirus VlP and serum obtained from a SARS convalescent patient.

To date, the most structurally complicated enveloped virus particle that has been used to generate VLP is influenza.VLPs for InfluenzaA H9N2 and H3N2 have been produced by other groups.PThese studies have shown that expression of the major structural protein MI alone is sufficient result in the budding ofvirus-likevesicles from insect cells.? Also, co-expressionofMl with M2, HA and NA leadsto the assembly of influenzaVLP and MI-HA and MI-HA-NA VLPs confer protection from lethal challengewith the same type influenza A in mice. 60 • 61 VLP production was also successfully achieved by co-expressing HA, NA, MI and M2 from influenza virus A/Udorn/72 (H3N2) using a single recombinant baculovirus.P To date none ofthese influenza VLP havebeen tested in humans. However the potential that HA and NA could be incorporated directly into these VLP from circulating influenza strains without passage in tissue culture has particular advantage for the control of rapidly changing influenzaA virus.

In addition to the use ofVLPs as direct immunogens, the efficiency with which they stimulate cellular and humoral responses has made them prime candidates as carrier moleculesfor the deliveryof epieopes, DNA and smallmolecules targetingother diseases. Thishas been facilitated by the excellent structural information that is often available for virus particlesallowingrational designof vaccines where epitopes are exposedon the surfaceof the VLP. Manyof the VLPsthat have been developedas vaccines in their own right have also been tested as delivery systems for other molecules. It is not possible here to providea full accountof this approach, as the literature on deliveryand display usingVLPsis at leastaslargeas that on VLP production for direct immunization (for reviewseeref 62). Howeverit is necessary at least to introduce this important area ofVLP-based vaccine development.The use ofVLPs as carrier molecules for epitopes for other diseases isnot limited to thoseVLPsthat areformed from the capsids of economically significant viruses. The reasonthat manyVLPsmakeexcellent carriermolecules for the delivery of epitopes in vaccines is most likely because the particulate VLP structure is readily taken up into antigen presenting cells and thus is able to prime long lasting CTL responses in addition to antibody responses. 6 One approach that maybe of use to overcome this constraint would be to link foreign protein sequences to capsidproteins in such a way that they extend the N or C termini of the protein and extendeither insideor outside to particle/" Ofcourse,this isonly suitablewhereone or both termini ofthe protein are exposedon the insideor outside faceof the capsid. So far, there are no VLP that we are awareof that havefullyexploitedthe potential of this approach but it has been successfully employedfor other protein-basedparticulate structures that are similar to VLPs in their stimulation of B-cell and T-cell responses and requirement for complexprotein-protein interactions for particleassembly/"?'

Despite the accumulatedevidence of the potential ofVLPs as potent immunogensfor many viralsystems that wehavediscussed, thereremains someresistance to the VLPapproachasageneral vaccinationstrategyfor diseases causedby viruses. In part this is due to some high profiledisappointing resultsfor VLP vaccines in the earlystages of development, for example an ineffective earlyvaccine for HIV basedon TyVLPS.72 Thisexampleraises a point of caution for VLPvaccine designers. In general. VLPs stimulate efficient cellular and humoral immune responses but, as with anyvaccine, they relyon the long term host response to be effective. VLPsdesignedto work in immunocompromisedindividuals need to overcome the samechallenges to efficient immune responseas any other vaccine approach. The notion that VLPsare ineffective vaccines is clearly a myth that isexplodedby the imminent release of two new VLP-based HPV vaccines. Indeed, the accumulateddata from the fieldsuggests that VLPsare more effective thanmanyother types of subunit vaccines, becausethey are more conformationally authentic and are saferthanmanylive viruspreparationsbecause they are usually freeof viralgeneticmaterial.VLPproduction doesnot appear to be limited to anyone type of virus or virusfamily, nor is it significantly limited by the complexityofthe virus particle."

The use of insect cells as a protein expression system offers excitingopportunities for the synthesis of conformationally authentic VLPs that are formed from the intracellularassembly ofmultiple proteins expressed in the samecell. The advantage of this system overothers used for protein expression is its capacityfor industrial scale synthesis of largeand multiple proteins and the fact that insectcells are the natural replicationreservoirfor manypathogenicviruses. Thusthe basiccellularmachinerythat normallyprocesses the infectiousform of the virusispresent within the expression system and available to produce authentic VLPs.

Expression of hepatitis B virus core antigen gene in Saccharomyces cerevisiae: synthesis of two polypeptides translated from different initiation codons

The GAG precursor of simian immunodeficiency virus assembles into virus-like particles

Assembly and release of HIV-l precursor Pr55gag virus-like particles from recombinant baculovirus-infecred insect cells

Assembly of double-shelled, virus-like particles of bluetongue virus by the simultaneous expression of four structural proteins

Virus-like particles induce MHC class l-resrricted T-cell responses. Lessons learned from the hepatitis B small surface antigen

Priming of strong, broad and long-lived HIV type 1 p55gag-specific CD8+ cytotoxic T-cdls after administration of a virus-like particle vaccine in rhesus macaques

Immunization with hepatitis C virus-like particles protects mice from recombinant hepatitis C virus-vaccinia infection

Virus-like particles as immunogens

Large scale production and downstream processing of a recombinant porcine parvovirus vaccine

Expression of vaccinia recombinant HPV 16 L1 and L2 ORF proteins in epithelial cells is sufficient for assembly ofHPVvirion-like particles

Papillomavirus L1 major capsid protein self-assemblesinto virus-like particles that are highly immunogenic

Synthesis and assembly of virus-like particles of human papillomaviruses type 6 and type 16 in fission yeast Schizosaccharomyces pornbe

Nasal immunization of mice with human papillomavirus type 16 (HPV-16) virus-like particles or with the HPV-16 L1 gene elicits specific cytotoxic T lymphocytes in vaginal draining lymph nodes

HPV6b virus like particles are potent immunogens without adjuvant in man

A controlled trial of a human papillomavirus type 16 vaccine

A Phase 1 study of a recombinant viruslike particle vaccine against human papillomavirus type 11 in healthy adult volunteers

Safety and immunogenicity trial in adult volunteers of a human papillomavirus 16 L1 virus-like particle vaccine

Vaccination against human papillomavirus infection : a new paradigm in cervical cancer control

Efficacyof a bivalent L1 virus-like particle vaccine in prevention of infection with human papillomavirus types 16 and 18 in young women : a randornised controlled trial

Sustained efficacy up to 4.5 years of a bivalent L1 virus-like particle vaccine against human papillomavirus types 16 and 18: follow-up from a randomised control trial

Recomb inant vaccine for canine parvovirus in dogs

Production of porcine parvovirus empty capsids with high immunogenic activity

A novd recombinant virus-like particle vaccine for prevention of porcine parvovirus-induced reproductive failure

Oral immunization with recombinant Norwalk virus-like particles induces a systemic and mucosal immune response in mice

Recombinant Norwalk virus-like particles administered intranasally to mice induce systemic and mucosal (fecal and vaginal) immune responses

Recombinant Norwalk virus-like particles given orally to volunteers: phase I study

Humoral, mucosal and cdlular immune responses to oral Norwalk virus-like particles in volunteers

Expression and self-assembly of empty virus-likeparticles of hepatitis E virus

Oral administration of hepatitis E virus-like particles induces a systemic and mucosal immune response in mice

Protection of cynomolgus monkeys against HEV infection by oral administration of recombinant hepatitis E virus-like particles

Molecular cloning and expression of major structural protein VP1 of the human polyomavirus JC virus: formation of virus-like particles useful for immunological and therapeutic studies

Effect of dose and long-term storage on the immunogenicity of murine polyomavirus VPl virus-like particles

Formation of poliovirus-like particles by recombinant baculoviruses expressing the individual VPO,VP3 and VP1 proteins by comparison to particles derived from the expressed poliovirus polyprotein

Formation of virus-like particles when the polyprotein gene (segment A) of infectious bursal disease virus is expressed in insect cells

Synthesis of bluetongue virus (BTV) corelike particles by a recombinant baculovirus expressing the two major structural core proteins of BTV

High-level expression of five foreign genes by a single recombinant baculovirus

Development of baculovirus triple and quadruple expression vectors: coexpression of three or four bluetongue virus proteins and the synthesis of bluetongue virus-like particles in insect cells

Fields' Virology, Fourth Edition. Philadelphia : Lippincott Williams and Wilkins

Protective efficacyof virus-like particles for bluetongue disease

Long-lasting protection of sheep against bluetongue challenge afier vaccination with virus-like particles: evidence for homologou s and partial heterologous protection

Recombinant virus vaccine for bluetongue disease in sheep

Assembly of double-shelled rotavirusllke particles by simultaneous expression of recombinant VP6 and VP7 proteins

Heterotypic protection from rotavirus infection in mice vaccinated with virus-like particles

Biochemical and immunologic comparison of virus-like particles for a rotavirus subunit vaccine

Lactogenic antibody responses in cows vaccinated with recombinant bovine rotavirus-like particles (VLPs) of two serotypes or inactivated bovine rotavirus vaccines

Low titer maternal antibodies can both enhance and suppress B cell responses to a combined live attenuated human rotavirus and VLP-ISCOM vaccine

Immunogen icity and protective efficacyof rotavirus 2/6-virus-likeparticles produced by a dual baculovirusexpressionvector and administered intramuscularly, intranasally, or orally to mice

Rectal immunization with rotavirus virus-like particles induces systemic and mucosal humoral immune responses and protects mice against rotavirus infection

Inrrarectal immunization with rotavirus 2/6 virus-like particles indu ces an anti -roravirus immune response localized in the intestinal mucosa and protects against rotavirus infection in mice

Assembly of SIV virus-like particles containing envelope proteins using a baculovirus expression system

Hepat itis C virus structural proteins assemble into viruslike particles in insect cells

Formation of wild-type and chimeric influenza virus-like particles following simultaneous expression of only four structural proteins

Immunization with hepatitis C virus-like particles induces humoral and cellular immune responses in nonhuman primates

Efficient assembly and release of SARS coronavirus-like particles by a heterologous expression system

Assembly of human severe acute respiratory syndrome coronavirus-Iike particles

Production and characterization of simian-human immunodeficiency virus-like particles

Safety and immunogenicity of recombinant human immunodeficiency virus-like particles in rodents and rhesus macaques

Intranasal immunization with SIV virus-like particles (VLPs) elicits systemic and mucosal immunity

Hepatitis C virus-like particles induce virus-specific humoral and cellular immune responses in mice

Virus-like particle (VLP) vaccine conferred complete protection against a lethal influenza virus challenge

Influenza virus-like particles comprised of the HA. NA and Ml proteins of H9N2 influenza virus induce protective immune responses in BALBlc mice

Virus-like particles as vaccines and vessels for the delivery of small molecules

Bacterial carriers and virus-like-particlesas antigen delivery devices: role of dendritic cells in antigen presentation

Differential uptake and cross-presentation of human papillomavirus virus-like particles by dendritic cells and Langerhans cells

Recombinant parvovirus-like particles as an antigen carrier: a novel nonreplicative exogenous antigen to elicit protective antiviral cytotoxic Tvcells

Heterologous papillomavirus virus-like particles and human papillomavirus virus-like particle immune complexes activate human Langerhans cells

A recombinant virus-like particle system derived from parvovirus as an efficient antigen carrier to elicit a polarized Th1 immune response without adjuvant

Individual rotavirus-like particles containing 120 molecules of fluorescent protein are visible in living cells

Induction of protective anti-viral cyto toxic Tvcells by a tubular structure capable of carrying large foreign sequences

Virus-derived tubular structure displaying foreign sequences on the surface elicit CD4 + Th cell and protective humoral responses

Induction of human immunodeficiency virus type l -specific Tvcells by a bluetongue virus tubule-vectored vaccine prime-recombinant modified virus Ankara boost regimen

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

Efficient self-assembly of human papillomavirus type 16 Ll and Ll -L2 into virus-like particles

Expression, self-assembly and anti-genicity of the Norwalk virus capsid protein

Recombinant rabbit hemorrhagic disease virus capsid protein expressed in baculovirus self-assembles into viruslike particles and induces protection

Characterization of a recombinant human calicivirus capsid protein expressed in mammalian cells

Expression and self-assembly of Grimsby virus: anti-genic distinction from Norwalk and Mexico viruses

Characterization of capsid genes. expressed in the baculovirus system. of three new genetically distinct strains of "Norwalk-like viruses

Assembly of empty capsids by using baculovirus recombinants expressing human parvovirus B19 structural proteins

Production of mink enteritis parvovirus empty capsids by expression in a baculovirus vector system: a recombinant vaccine for mink enteritis parvovirus in mink

Chimeric virus-like panicle formation of adeno-associared virus

Immunogenic and protective properties of chicken anaemia virus proteins expressed by baculovirus

Simultaneous expression of recombinant baculovirusencoded chicken anaemia virus (CAV) proteins VPl and VP2 is required for formation of the CAV-specific neutralizing epitope

An experimental model for post-weaning multisystemic wasting syndrome (PMWS) in growing piglets

Characterization of the recombinant proteins of porcine circovirus type2 field isolate expressed in the baculovirus system

Purification and characterization ofvirus-like particles and pentamers produced by the expression of SV40 capsid proteins in insect cells

Self-assemblyof the JC virus major capsid protein . VPl. expressed in insect cells

Formation of poliovirus-like particles by recombinant baculoviruses expressing the individual VPO. VP3 and VP 1 proteins by comparison to panicles derived from the expressed poliovirus polyprocein

Characterization of virus-like panicles produced by the expression of rotavirus capsid proteins in insect cells

The protease and gag gene products of the human immunodeficiency virus: authentic cleavage and post-translational modification in an insect cell expression system

Characterization of virus-like panicles produced by a recombinant baculovirus containing the gag gene of the bovine immunodeficiency-like virus

Analyses of the requirements for the synthesis of virus-like particles by feline immunodeficiency virus gag using baculovirus vectors

Expression of feline leukaemia virus gp85 and gag proteins and assembly into virus-like particles using the baculovirus expression vector system

Immune response to recombinant visna virus Gag and Env precursor proteins synthesized in insect cells

Bovine leukemia virus Gag panicle assembly in insect cells: Formation of chimeric particles by domain-switched leukemia/lentitivirus Gag polyprotein

PR domain of rous sarcoma virus Gag causes an assembly/budding defect in insect cells

Synthesis of Newcastle disease virus (NDV)-like envelopes in insect cells infected with a recombinant baculovirus expressing the haemagglutinin-neuraminidase of NDV

Nucleocapsid-and virus-like particles assemble in cells infected with recombinant baculoviruses or vaccinia viruses expressing the M and the S segments of Hanraan virus

Expression of ORF Al of infectious bursal disease virus results in the formation of virus-like particles

Structuredcpendent efficacy of infectious bursal disease virus (IBDV) recombinant vaccines