1-s2.0-s0168165604003025-main

Journal of Biotechnology 113 (2004) 1532

Biotechnology and vaccines: application or bie, B

za, V, 53100

Received 9 October 2003; received in revised form 9 March 2004; accepted 19 March 2004

Abstract

Since itsconventionahas failed tobiotechnologclinical diagit is possiblewhich has band review tthe identifictechnologies 2004 Else

Keywords: V

1. Introdu

Approacenced remMost of th

Correspofax: +39-0577

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0168-1656/$doi:10.1016/jintroduction, vaccinology has been very effective in preventing infectious diseases. However, in several cases, thel approach to identify protective antigens, based on biochemical, immunological and microbiological methods,

deliver successful vaccine candidates against major bacterial pathogens. The recent development of powerfulical tools applied to genome-based approaches has revolutionized vaccine development, biological research and

nostics. The availability of a genome provides an inclusive virtual catalogue of all the potential antigens from whichto select the molecules that are likely to be more effective. Here, we describe the use of reverse vaccinology,

een successful in the identification of potential vaccines candidates against Neisseria meningitidis serogroup Bhe use of functional genomics approaches as DNA microarrays, proteomics and comparative genome analysis foration of virulence factors and novel vaccine candidates. In addition, we describe the potential of these powerfulin understanding the pathogenesis of various bacteria.

vier B.V. All rights reserved.

accines; Genomics; Reverse vaccinology; Microarray; Proteomics

ction

hes to vaccine development have experi-arkable progress during the last century.e vaccines currently available were gen-

nding author. Tel.: +39-0577-243414;-243564.ress: rino [email protected] (R. Rappuoli).

erated a long time ago and are based on killed orlive-attenuated microorganism, on toxins detoxifiedby chemical treatment, on purified antigens and onpolysaccharide conjugated to proteins. The knowl-edge of the pathogenesis of many microorganisms, theidentification of the main virulence factors and thecharacterization of the immune response after infec-tion have been fundamental for the design of second-generation vaccines mainly based on highly purified

see front matter 2004 Elsevier B.V. All rights reserved..jbiotec.2004.03.024Neisseria meningitidis and otheDavide Serruto, Jeannette Adu-Bob

Rino Rappuoli, Mariagrazia PizIRIS, Chiron Vaccines,Via Fiorentina 1f functional genomics toacterial pathogensarbara Capecchi,ega MasignaniSiena, Italy

16 D. Serruto et al. / Journal of Biotechnology 113 (2004) 1532

antigenic components (Rappuoli and Del Giudice,1999).

The development of prophylactic vaccines to pre-vent diseasimprovemeDevastatingbeen totallythere are mficacious foFor these rways to dis

Recentlygained newis clear thaover; secontive of all mHowever, mnew technoical barrierThe genomtechnologic

The Woleased a rephighlights thealth (WH

A pionepromisingveloping co2002). In thgories wascines again

Modernused to prodgens. Thepure surfac1990). Thedetella perThe latter vfunction stsis toxin thtered antigenovel approfor vaccinetwo decadestudied froimmunologvirulence tThis metho

of most new vaccines currently available or in clinicaldevelopment.

Among them, the recombinant vaccine againstlus anen PAof theand wced b

to indA-bas, undenes orveral

arried. An eable o

withhe liverecom

ticallyvaccinted; h

nd repalderrae v

maticachol

technnother

vacc

gic asurfac

protec998). Aed byved fpA-bag the996).nto ace prote).rthermevaluas Mophil

), Stre) and fes has made an essential contribution to thent of human health during the 20th century.diseases such as smallpox and polio haveor almost completely eradicated. However,any infectious diseases still waiting for ef-rmulations and many emerging pathogens.easons, novel vaccines together with newcover and produce them are needed., vaccine research and development hasimpetus for a number of reasons. First, it

t our fight against bacterial infection is notdly, vaccines represent the most cost effec-edical interventions (Rappuoli et al., 2002).ost importantly, the rapid development of

logies has allowed to overcome technolog-s that used to limit vaccine development.ic revolution is one of the most importantal advances.

rld Health Organization (WHO) recently re-ort titled Genomics and World Health thathe potential of genomics to improve globalO, 2002).

er study by Daar et al. identified the 10 mostbiotechnologies for improving health in de-untries in the next 510 years (Daar et al.,is study, one of the most highly rated cate-

Recombinant technologies to develop vac-st infectious diseases.recombinant DNA technology has beenuce subunit vaccines based on specific anti-

first of these new vaccines was the highlye antigen of the hepatitis B virus (Andre,second was the subunit vaccine againstBor-tussis containing three highly pure proteins.accine also pioneered the use of structure

udies to produce genetically altered pertus-at lacked toxicity but maintained an unal-nic conformation (Pizza et al., 1989). Theseaches led to the development of a paradigmresearch, which has persisted for the lasts. In this approach, the microorganism ism the point of view of pathogenicity andy in order to identify factors involved in

hat may be suitable as vaccine candidates.dology has been used for the development

BacilantignentLF),produableby Ptionsvacci

Seing ccinesavailbinedand twithzymaThistoleraing aand Ccholeenzyentirenetic

Abasedetiolooutersess

al., 1velopapproof Osamon

al., 1ing iof th2002

FubeingsuchHaem20032003thracis consists of the use of the protective, which corresponds to the beta subcompo-anthrax edema factor or lethal factor (EF orhich is recognized as the major immunogeny the bacterium (Turnbull, 1991). Althoughuce good protective immunity, vaccinationed vaccines requires multiple immuniza-rlying the need to develop more efficaciousalternative vaccination regimens.

recombinant-based approaches are also be-out for the development of new cholera vac-xample is represented by the commerciallyral vaccines, such as the whole cell com-a non-toxic form of the B subunit (WCBS)-attenuatedVibrio cholerae strain, producedbinant techniques and not expressing the en-active subunit of the cholera toxin (CtxA).

e is safe, immunogenic and generally wellowever, the protective effect is not long last-eated booster doses are recommended (Ryanwood, 2000). Additional live-attenuated V.accines are based upon the removal of thelly active subunit of cholera toxin or of the

era toxin genetic element by molecular ge-iques and are in various stages of analysis.

interesting example of a single antigen-ine is that against Borrelia burgdorferi, thegent of lyme borreliosis. In this case, thee lipoprotein OspA has been shown to pos-tive activity against the disease (Steere etn OspA vaccine, LYMErix, which was de-SmithKline Beecham, has been recently

or human use; however, a major deficiencysed vaccines is the variability of the antigen

various species of B. burgdorferi (Wilske etFor this reason, alternative approaches tak-count the use of single conserved portionsin are currently being pursued (Luft et al.,

ore, protein-based approaches are alsoated for other important human pathogens,oraxella catarrhalis and non-typeable

us inuenzae (McMichael and Green,ptococcus pneumoniae (Swiatlo and Ware,or some viruses, such as hepatitis C (HCV)

D. Serruto et al. / Journal of Biotechnology 113 (2004) 1532 17

(Hsu et al., 1999), human immunodeficiency virus(HIV) (Amara and Robinson, 2002), human papillomavirus (Rohan et al., 2003) and EpsteinBarr virus(Finerty et

Finally,ral and bacombinantGuzman, 2is now possgenes to invectors andimmunogetors is thatimmune syand to the r

Other twthe study bgenomes tonew antimdrug targetions.

Automathe availabolutionizeding the neSander, 19complete gobtained ingun sequecessfully u(TIGR) inof H. inulast few yegrown consgenome dathe bacterigenome seganisms araround theat http://wpanel of bthe pathogetherefore o

Bioinfoamount ofquences (gbe used totures such

ubility. Moreover, a putative function can be assignedto each open reading frame (ORF) on the basis of ahomology to known proteins. Sophisticated computer

ams af newto cho

omplehes acs. Tsis ofologym (proresismepr

onveninolog

e conased oatoryolatedologicformmunecases

ds toto no

, the aced i

oy maen andgensctive ae reve

ntage ome rehe patble torevers

from tgenoman benovely (F

ere, wh hasal., 1994).it is worth mentioning the use of live vi-

cterial vectors as delivery systems for re-vaccines (Stephenson, 2001; Medina and

001). On the basis of genetic information, itible to select a priori the bacterial and viral

activate to improve the safety profile of liveto identify the potential antigens to use as

ns. The major advantage in using live vec-the heterologous antigen is presented to thestem by the host in its native conformationight compartment.o most highly rated categories presented iny Daar et al. were Sequencing pathogenunderstand their biology and to identify

icrobials and Bioinformatics to identifyts and to examine pathogenhost interac-

ted DNA sequence analysis, together withility of new bioinformatic tools, has rev-the field of biology and medicine, open-

w era of genomic science (Andrade and97; Brutlag, 1998; Fraser et al., 2000). Theenome sequence of a bacterium can bea brief period of time using the shot-

ncing strategy. This technique was suc-sed at The Institute for Genomic Research1995, to determine the genome sequenceenzae (Fleischmann et al., 1995). In thears, the number of available genomes hasiderably (Fig. 1). Today, a quick look at thetabases reveals the level of knowledge onal world: in addition to the 156 completedquences, more than 400 other microor-e being sequenced in various laboratoriesworld (GOLD Genomes OnLine databaseit.integratedgenomics.com/GOLD/). Thisacterial genomes already covers most ofns impacting heavily on human health and

f interest for vaccine researchers.rmatics is essential to interpret the immenseinformation contained in whole genome se-enomic mining). A variety of software canassign gene functions and predict key fea-as topology, molecular weight, pI and sol-

progrtion osible

Cproacnomianalytechnganistrophgeno

2. Cvacc

Thare blaborare iscrobipurean immanyIt neecablecases

produemplantigpathoprote

Thadvagenothat tpossiin athanogy,that cThisnolog

Hproacre also available to predict cellular localiza-ly identified ORFs, so that it becomes pos-ose potentially surface-exposed proteins.mentary to in silico antigen discovery ap-re strategies referred to as Functional Ge-hese approaches include the large-scalegene transcription, using DNA microarray, the whole set of proteins encoded by an or-oteomics) using two-dimensional gel elec-and mass spectrometry and the comparativeoteome technologies.

tional vaccinology versus reversey

ventional approaches to produce vaccinesn the cultivation of the microorganism inconditions from which single componentsindividually by using biochemical and mi-al methods. Each antigen is produced inand finally tested for its ability to induceresponse. However, although successful in, this approach presents several limitations.

grow the pathogen in vitro, so it is not appli-n-cultivable microorganisms, and in manyntigens expressed during infection are notn laboratory conditions. This method canny years to identify a protective and usefulhas failed to provide a vaccine against those

that did not have obvious immunodominantntigens (i.e. capsule or toxins).rse approach to vaccine development takesf the genome sequence of the pathogen. The

presents, virtually, a list of all the proteinshogen can express at any time. It becomeschoose potentially surface-exposed proteinse manner, starting from the genome ratherhe microorganism. In the field of vaccinol-ics represent a fantastic reservoir of genesscreened and tested as vaccine candidates.approach has been coined reverse vacci-

ig. 2) (Rappuoli, 2000, 2001).e describe how a reverse vaccinology ap-been successful to identify novel potential


Fig. 1. Schematic graph showing a representative list of available bacterial genomes increasing in the last years. (Four different websites were used as sources: the TIGR web site, http://www.tigr.org; the Sanger web site, http://www.sanger.ac.uk/; the NCBI website, http://www.ncbi.nlm.nih.gov/PMGifs/Genomes/micr.html and the GOLD Genomes OnLine database at http://wit.integratedgenomics.com/GOLD/.)


Fi

vaccine canseria menincation of fu

3. ReverseMeningoco

N. menisepsis, twoand youngbacteriumnasopharynpopulationsignificantthe epithelsepticemiacross the bcausing me

The incfrom 13or developg. 2. Comparison of methodologies and time between conventional and rev

didates against the human pathogen Neis-gitidis serogroup B and illustrate the appli-nctional genomics to vaccine research.

vaccinology applied to group Bccus

ngitidis is the major cause of meningitis anddevastating diseases that can kill children

adults. Meningococcus is a Gram-negativethat colonizes asymptomatically the upperx tract of about 515% of the human

. However, for reasons yet unknown, in anumber of cases, the bacterium can traverseium and reach the bloodstream causing. From the blood, Meningococcus is able tolood brain barrier and infect the meninges,ningitis.idence of meningococcal disease rangesto 1025/100,000 cases in industrializeding countries, respectively, and despite se-

vere antibihigh.

N.meninthe basis omore thancaused by fiEfficaciouspolysacchacurrently aglycoconjulicensed. Tficacy of 97venting moal., 2001).other serog

The critfor which tcannot beMenB capsacid, a commalian tissself-antigenerse vaccinology to vaccine development.

otic therapies, the mortality rates remain

gitidis can be classified in 13 serogroups onf their polysaccharides capsule. However,95% of total cases of invasive disease areve major serogroups: A, B, C, Y and W135.vaccines composed from purified capsule

rides of serogroups A, C, Y and W135 arevailable. More recently, a new generation ofgate vaccine against serogroup C has beenhis vaccine, tested in UK, has shown an ef-% in adolescents and 92% in toddlers, pre-re than 500 cases in one year (Ramsay etA similar approach will be extended to theroups A, Y and W135.ical point remains with serogroup B (MenB)he polysaccharide-based vaccine approachused. In fact, the major component of theule is the(28)-linkedN-acetylneuraminicmon carbohydrate present also in the mam-ue. Therefore, this polysaccharide, being a, is poorly immunogenic and may elicit au-


toimmunity. An alternative approach to vaccine devel-opment is based on surface-exposed proteins containedin the outer membrane vesicles (OMVs). However, dueto the highantigens pshown to ethe homoloal., 1999; d

Forty yeto vaccine rvaccine agacauses fortries. To ovnamed rev(Pizza et al.the virulention with Tgenome coage ofG +Ccodes for 21biological rwith knownnot a predi

Based ogens are motherefore acine, the fuics tools infor putative

All theputer progsignal pepttative hydroidentify lipteins withother bactelowed the iclassified ocreted or ou(20%), perproteins (3ogy (6%). Acloned in Eas N-termiterminal hirecombinanto immunizwith severa

in Western blot on total cell lysate and outer mem-brane preparation to confirm that each protein was re-ally expressed in vivo and localized in the outer mem-

. ELIere pee exp

for t, whicmans.ive inive in tandid

es.s menin thence vs. Fo, Por

ctiont, these vac

nce vs, inca andR fro

sequenrify ths. Suronlyprotentitywere a

illinglemenlloweens thing aterestides anibed plogieTo v

e pathins haical af themthe laath ofsequence and antigenic variability of theresent in the OMVs, these vaccines havelicit protective antibodies but only againstgous strain (Bjune et al., 1991; Tappero ete Moraes et al., 1992).ars of studies based on classical approachesesearch have failed to provide an efficaciousinst MenB that remains one of the principle

bacterial meningitis in industrialized coun-ercome the latter obstacle, the new approacherse vaccinology was applied to MenB, 2000). To this end, the complete genome oft strain MC58 was sequenced in collabora-IGR using the shotgun strategy. The MenBnsists of 2,272,352 base pairs with an aver-

content of 51.5%. The 83% of the genome58 ORFs. Out of these, 1158 have a putativeole assigned on the basis of their similarityproteins, whereas the remaining 1000 have

cted function (Tettelin et al., 2000).n the concept that surface-exposed anti-re susceptible to antibodies recognition andre the most suitable candidates for a vac-ll genome was screened using bioinformat-order to select open reading frames codingsurface-exposed or secreted proteins.

putative ORFs were analyzed using com-rams as PSORT or SignalP, to predict theide sequences, TMPRED to identify the pu-phobic membrane regions and MOTIFS tooproteins. Finally, ORFs coding for pro-homology to known virulence factors ofria were also included. This screening al-

dentification of 600 putative ORFs that weren the basis of their predicted features: se-ter membrane proteins (13%), lipoproteins

iplasmic proteins (27%), inner membrane4%) and proteins with interesting homol-

ll these ORFs were amplified by PCR andscherichia coli, in order to express them

nal glutathione-S-transferase (GST) or C-stidine-tag fusion. Three hundred and fiftyt proteins were expressed, purified and usede mice. The sera obtained were then testedl assays. First of all, sera were analyzed

braneria wof thtestedtivityin hupositpositfew cstudi

AfacesequestrainMenBproteof tharever

sequestraintamicby PCTheto vealleletifiedotherof idegensrial kcompogy aantigvelop

Inprovidescrhomoteria.in thprotechemtion o

Inthe pSA and FACS analysis on whole-cell bacte-rformed to verify the surface-localization

ressed proteins. Finally, all the sera wereheir complement-mediated bactericidal ac-h is known to correlate with the protectionFrom this screening, 91 proteins resulted

at least one assay, and out of them, 28 werehe bactericidal assay (Fig. 3). Among these,ates were selected and subjected to further

tioned before, one of the main problems todesign of a vaccine against MenB is the

ariability of the antigens among differentr example, the most abundant antigen ofA, is extremely variable and able to conferonly against the homologous strain. In viewnine best vaccine candidates selected by thecinology approach were analyzed for theirariability using a representative panel of 31lusive of N. meningitidis, N. cinerea, N. lac-N. gonorrhoeae. Each gene was amplifiedm all the 31 selected strains and sequenced.ces were subjected to multiple alignmentse level of homology among the differentprisingly, hypervirulent regions were iden-in the case of two antigens, whereas all theins were highly conserved, with percentageof about 99%. Finally, these conserved anti-ble to induce complement-mediated bacte-in a subset of strains for which a suitablet source was available. Reverse vaccinol-d the identification in a few years of manyat now could be considered as basis for de-vaccine against MenB.ngly, the availability of the entire genomeinexhaustible source of unknown and un-

roteins, several of them sharing attractives with known virulence factors of other bac-erify whether they could have a role alsoogenesis of Meningococcus, some of theseve been further characterized from the bio-nd functional point of view. A brief descrip-

is reported in Table 1.st few years, several groups have followedpioneer work on MenB, utilizing the ap-


Fig. 3. N. meningitidis serogroup B as an example of reverse vaccinology: (a) use of different bioinformatic software to analyze the genome; (b)identification of potential vaccine candidates and percentage distribution according to their topological features; (c) selected ORFs are amplified,cloned in expression vectors, purified and used to immunize mice; and (d) mice immune sera are analyzed using FACS to verify whether theantigens are expressed and surface-exposed; the bactericidal assay is used to evaluate the complement-mediated bacterial killing activity ofantibodies.

Table 1Description of some of the novel MenB antigens identified

N. meningitidis antigen Description Reference

GNA33 Membrane-bound lytic transglycosylase (MltA) of N. meningitidis Granoff et al., 2001; Jennings et al., 2002;Adu-Bobie et al., 2004

GNA992 A putative adhesin homologue to Hsf and Hia proteins Scarselli et al., 2001NadA A new adhesin vaccine candidate Comanducci et al., 2002GNA1870 Surface-exposed lipoprotein as vaccine candidate Masignani et al., 2003bApp An autotransporter adhesin with autocatalytic serine protease activity Serruto et al., 2003NarE A novel ADP-ribosylating enzyme Masignani et al., 2003a


proach of reverse vaccinology and functional genomics(microarray, proteomics and comparative analysis) toidentify new vaccine candidates. Bacteria that havebeen studieB. anthracStaphylocoListeria molosis.A comproaches de

4. DNA m

DNA mveloped gepowerful tocomplete sBotstein, 11999; Lockin the techsion profilbiologicaldiscovery.

Microarapplicationgenotypingexpressionlevels. Theis to estabdifferentlythat of mullyzed in terand co-regcellbiologunderstandthat are reset al., 200to identifyresponse toto evaluateways. Morescriptome otissues and,scriptionalray technolextensivelyreviews (S2000; Kato

nik, 2003). In this section, we describe some of therecent studies that provide forceful proof that themicroarray technology allows to examine the dynam-

f a wogatinsing S.genes

regulase (Dud thatoleraecriptiond, co, 2002hole-ylorirates ahase-t003). T

were

micrntini egen (Ge como beiexpregensand/oNA palyzeds undeere arctionteractonellacellshial e000),(Ichikwith a001),atis (

se to i), globto Shioweve

the hray ted include several human pathogens such asis, Chlamydia pneumoniae, S. pneumonia,ccus aureus, Porphyromonas gingivalis,nocytogenes and Mycobacterium tubercu-prehensive list of the different genomic ap-scribed in this review is reported in Table 2.

icroarray technology

icroarray (or microchips) is a recently de-nomic technology and is one of the mostols for the study of the transcriptome, the

et of transcripts of an organism (Brown and999; Cheung et al., 1999; Lipshutz et al.,hart and Winzeler, 2000). Recent advancesnology of massive parallel gene expres-

ing using microarrays are revolutionizingresearch, clinical diagnostics and vaccine

ray technology can be used for severals including gene expression profiling,and DNA sequencing. As a result, gene

array data can be analyzed on at least threefirst level is that of single genes; the aim

lish whether each isolated gene behavesin different conditions. The second level istiple genes, where clusters of genes are ana-ms of common functionalities, interactionsulation. Finally, the third level is that ofy metabolism pathways, where the aim is tothe underlying gene and protein networksponsible for the various patterns (Hatfield

3). Researchers are using this technologygenes that are differentially expressed inalteration in environmental parameters and

mutations in regulatory and metabolic path-over, another purpose is to capture the tran-f bacteria growing within infected cells andas a result, to disclose the host-adapted tran-reaction. The applications of DNA microar-ogy in all these biological fields have beendescribed elsewhere by several excellent

choolnik, 2002b; Cummings and Relman,-Maeda et al., 2001; Conway and School-

ics ointerr

UtifiedtwosponstrateV. chtranstant aet al.the wter pillustlog pal., 2MenBDNAGrifapatho

This alsgenepathoturesfor Ris angene

Thinterathe inSalmcyticbroncal., 2nosa

cellsal., 2chomspon2003cells

Hfromcroarhole biological system by simultaneouslyg the expression of thousands of genes.aureusGeneChip, Dunman et al. have iden-that are regulated by agr and/or SarA, the

tors of the microorganisms virulence re-nman et al., 2001). Mekalanos et al. demon-quorum-sensing regulators are involved invirulence. Gene arrays were used to profilen in the wild-type strain and in the luxOmu-nsequently, to define the LuxO regulon (Zhu). In a recent study, Falkow et al. investigatedgenome expression profiling of Helicobac-grown in vitro. This time course analysismajor switch in gene expression at the late

o-stationary phase transition (Thompson ethe iron-activated and -repressed genes ofN.identified by transcriptome analysis: using

oarray, computational and in vitro studies,t al. defined the Fur regulon of this humanrifantini et al., 2003).

plex interaction between host and pathogenng explored using microarrays. Virulencession can be monitored by growing the

in the appropriate in vivo models (cell cul-r animals) and, after recovering the bacteriareparation and labelling, the gene activityand compared with the expression of the

r in vitro conditions.e several studies focused on hostpathogen

s using DNA microarrays and they includeions between intestinal epithelial cells and(Eckmann et al., 2000), human promyelo-

and L. monocytogenes (Cohen et al., 2000),pithelial cells and B. pertussis (Belcher etepithelial cells and Pseudomonas aerugi-awa et al., 2000), mouse gastric epithelialnd without exposure to H. pylori (Mills etHeLa cells infected with Chlamydia tra-

Xia et al., 2003), human macrophage re-nfection with M. tuberculosis (Wang et al.,al response of human intestinal epithelialgella exneri invasion (Pedron et al., 2003).r, these studies consider gene activationost perspective. Recently, the DNA mi-chnology has been applied to study the


Table 2Examples of application of functional genomic approaches to bacterial pathogens

Bacterium Approaches References

Bacillus anthracis Reverse vaccinology Ariel et al., 2002Comparative genome analysis Read et al., 2003Serological proteome analysis Ariel et al., 2003

Bordetella pertussis Microarray Belcher et al., 2000Campylobacter jejuni Comparative genome analysis Pearson et al., 2003Chlamydia pneumoniae Proteomics/reverse vaccinology Montigiani et al., 2002

Chlamydia trachomatis Microarray Xia et al., 2003Microarray Belland et al., 2003

Escherichia coli Comparative genome analysis Dobrindt et al., 2003Haemophilus inuenzae Proteomics Langen et al., 2000; Thoren et al., 2002Helicobacter felis Microarray Mueller et al., 2003Helicobacter pylori Microarray Mills et al., 2001

Microarray Thompson et al., 2003Comparative genome analysis Salama et al., 2000Comparative proteome analysis Jungblut et al., 2000; Govorun et al. 2003Serological proteome analysis Utt et al., 2002; Baik et al., 2004

Listeria monocytogenes Microarray Cohen et al., 2000Comparative genome analysis Glaser et al., 2001; Buchrieser et al., 2003

Mycobacterium tuberculosis Microarray Wang et al., 2003Microarray Schnappinger et al., 2003; Wilson et al., 1999STM Camacho et al., 1999Comparative genome analysis Cockle et al., 2002Comparative proteome analysis Jungblut et al., 1999; Mattow et al., 2001

Mycoplasma pulmonis DNA vaccination Barry et al., 1995

Neisseria meningitidis Reverse vaccinology Pizza et al., 2000Microarray Grifantini et al., 2002a,b, 2003; Kurz et al., 2003STM Sun et al., 2000Whole genome expression library Pelicic et al., 2000Comparative genome analysis Perrin et al., 2002

Porphyromonas gingivalis Reverse vaccinology Ross et al., 2001

Pseudomonas aeruginosa Microarray Ichikawa et al., 2000IVET Wang et al., 1996a,b

Salmonella typhimurium Microarray Eckmann et al., 2000STM Hensel et al., 1995IVET Mahan et al., 1995DFI Valdivia and Falkow, 1996, 1997a

Shigella exneri Microarray Pedron et al., 2003Staphylococcus aureus Microarray Dunman et al., 2001

STM Coulter et al., 1998; Mei et al., 1997IVET Lowe et al., 1998Genomic peptide libraries Etz et al., 2002Serological proteome analysis Vytvytska et al., 2002

Streptococcus agalactiae Comparative genome analysis Tettelin et al., 2002Proteomics Hughes et al., 2002

Streptococcus pneumoniae Reverse vaccinology Wizemann et al., 2001STM Polissi et al., 1998


Table 2 (Continued )Bacterium Approaches References

Comparative genome analysis Oggioni and Pozzi, 2001

Streptococcus is

Vibrio cholera

Yersinia enter

Yersinia pesti is

Note: microa from thbacterial gene

gene expredifferent st

Bellandscriptionaltrachomatianalyze thethroughoutnew findingtrol the diffof the hostproducts thcases, havekaryotic ordiae may hhave used tbiology. Thtechnologylight into tchlamydiaetools for thferently excovered wi(Belland et

Anothertranscriptiomacrophagculosis is amacrophagof phagosodevelopmecollaboratothis pathogferently exsis in nav

withstudyanscrionmenmes ancretedFinallculosith to ionmenicroarand thlso toes andne exassfulas ne

ngitidngococandidto stungitidpyogenes Comparative genome analys

e MicroarraySTMIVET

ocolitica STMIVET

s Comparative genome analys

rray studies indicated with an asterisk () consider gene activationexpression profile.

ssion profile of human pathogens duringages of infection.et al. have characterized the genomic tran-profiling of the developmental cycle of C.s. In this work, microarrays were used to

temporal expression of chlamydial genesthe life cycle. This approach has provideds about chlamydial gene products that con-erentiation stages and determine the naturepathogen interaction. These genes encodeat are chlamydial-specific and in certainphylogenetic signatures that suggest eu-

igin. The authors hypothesize that Chlamy-ave acquired these genes from the host andhem functionally to define their distinctiveis work demonstrated also that microarrayis a powerful and indispensable tool to shed

he complex interactions between host andbecause there are not genetic exchange

e bacteria; consequently, many of the dif-pressed genes would likely remain undis-thout the application of microarray analysis

paredThethe trenvirenzyof seiron.tubergrowenvir

Mderstbut adidat

Osucce

wellmeniMenicineusedmenial., 2003).particularly informative study defined thenal adaptation of M. tuberculosis withines (Schnappinger et al., 2003). M. tuber-ble at adapting to long-term residence ine phagosomes because it blocks maturationmes into phagolysosomes by controlling thent of these compartments. Schoolnik andrs captured the global expression profile ofen and identified the genes that are dif-pressed by intraphagosomal M. tuberculo-e and INF--activated macrophages, com-

2002b). RNadherent bamicroarrayamplified Mterial adhesion of appregulated aseven were

pending othe regulatjor categortalk genes,Smoot et al., 2002

Zhu et al., 2002Chiang and Mekalanos, 1998Camilli and Mekalanos, 1995

Darwin and Miller, 1999Young and Miller, 1997

Hinchliffe et al., 2003

e host perspective; all the other papers analyzed the

bacteria grown in standard broth culture.demonstrated that the bacterium modifiesptome in order to adapt to the phagosomalt by the induction of fatty acid-degradingd DNA repair proteins and the productionsiderophores to facilitate the acquisition ofy, the authors used expression profiles of M.s exposed to various in vitro conditions ofsolate situations that mimic the phagosomalt (Schnappinger et al., 2003).

rays provide a powerful tool, not only to un-e regulation of gene expression in bacteria,discover new virulence genes, vaccine can-drug targets.

mple where microarray technology has beento identify potential vaccine candidates, asw virulence genes, is in the case of N.is. In order to understand the pathology ofccus and for the identification of new vac-ates, DNA microarray technology has beendy gene regulation after interaction of N.is to human epithelial cells (Grifantini et al.,

As were isolated from adherent and non-cteria and comparatively analyzed on DNAs carrying the entire collection of PCR-

enB genes. The authors found that bac-sion to epithelial cells altered the expres-roximately 350 genes: 189 genes were up-nd 151 genes were downregulated whileeither upregulated or downregulated de-

n the time point of infection. Most ofed genes can be grouped into five ma-ies: adhesion genes, hostpathogen cross-

amino acids and selenocysteine biosyn-


thesis genes, DNA metabolism genes and hypothet-ical genes. Moreover, of the 12 adhesion-inducedsurface-exposed antigens identified, five were ableto induce bstudy showto identifyment othervaccinolog

In an indof N. meninof three kewas isolateserum as w

endothelialrange of suvivo conditcandidatescoccal dise

The firssitology arplete genomrecently puvelopmentsequence a2002; Longcatalogue tparasitic lifthe upreguljects. Selecevaluated a

Recentlexpressiondrugs andDNA micrM. tubercuexpressionniazid. Thelog phase bencode promode of acmediate prquences ofpoints up hto the drugganism toaction shouinates the apathway (S

The understanding of the protective mechanism me-diated by each vaccine is an essential prerequisite to de-sign new rationally based vaccines. In a recent work,

w andnd imanismfHelapprowill hction

recen

es in Damoun

discobecaufluenbe int

have liproteinys hav). Conoxicit

vestig

ene exo idenne deted ton infeccines faciltaggeden (Hcted tsuch

fic shozationnts cave pastial fothis a

ificatictivenes. Mr infefor inactericidal antibodies. In conclusion, thiss that DNA microarray technology is ablepotential vaccine candidates and comple-genome mining methods such as reverse

y.ependent study, the transcriptional changesgitidis were investigated in a model systemy steps of meningococcal infection. RNAd from Meningococci incubated in humanell as adherent to human epithelial andcells. The authors discovered that a widerface proteins which are induced under inions. These antigens could represent novelfor a protein-based vaccine for meningo-ases (Kurz et al., 2003).t applications of DNA microarray in para-e in place (Rathod et al., 2002). The com-

e sequence ofPlasmodium falciparumwasblished and systematic approaches to the de-of vaccines based on the completed genomere already in planning stages (Gardner et al.,

and Hoffman, 2002). One approach is tohe expression of proteins at each stage of theecycle using this technology and screeningated proteins using sera from immune sub-ted genes can then be cloned, expressed ands vaccine components in challenge studies.y, a pioneer work showed how microarrayprofiling could be used to discover new

their mode of action. Wilson et al. used aoarray containing 97% of the ORFs of thelosis genome to examine changes in the genein response to the antituberculous drug iso-y reported that isoniazid treatment of mid-acterial cultures induced several genes thatteins physiologically relevant to the drugstion. Other genes were induced and likelyocesses that are linked to the toxic conse-the drug (Wilson et al., 1999). This study

ow gene expression analysis can contributediscovery process: exposure of a microor-

a drug or compound of unknown mode ofld elicit an expression profile that incrim-ffected pathway and even the target in thechoolnik, 2002b).

Falkoing amechcine oThisgies,prote

Invanc

ablegeneever,

are inmustdataflectalwa1999and t

5. In

Gder tvaccimulaing ain vabut itureHoldsubjesis inspecidridimutasurviessen

usingidentproduvaccitial fodatescollaborators used gene expression profil-munohistochemical analysis to unravel theof protection of a whole-cell sonicate vac-

icobacter felis in mice (Mueller et al., 2003).ach, applied to other immunization strate-elp to better understand the mechanism ofof several vaccine formulations.t years, there have been enormous ad-NA microarray technology and a remark-t of literature supporting its central role in

very, vaccine and drug development. How-se the results of pathogen gene expressionced by the model system used, such resultserpreted cautiously. In addition, expressionmitations because mRNA levels may not re-

levels, and expression of a protein may note a pathological consequence (Gygi et al.,sequently, traditional biological, pathologyy studies remain necessary.

ating gene expression in vivo

pression in vivo has been exploited in or-tify virulence genes, which is a key step insign. A variety of methods have been for-isolate genes that are specially induced dur-ction. One of the recent technologies useddesign, which does not strictly depend onitated by genome sequencing, is the signa-

mutagenesis (STM) developed by Davidensel et al., 1995). A bacterial pathogen iso random transposon-mediated mutagene-a way that each mutant is tagged with art DNA sequence tag. Comparison, by hy-

, of the tags found in arrays representing allpable of growing in vitro with mutants thatsage through an animal host identifies genesr the infectious process. The advantage ofpproach is that the technique allows for theon of attenuated mutants that fail to cause ainfection and therefore may be used as liveoreover, proteins identified as being essen-

ction or disease are likely to be good candi-clusion in subunit vaccines. STM has been


successfully used to discover virulence genes from avariety of bacterial species including M. tuberculosis(Camacho et al., 1999), S. aureus (Coulter et al., 1998;Mei et al.,al., 1995),Yersinia enpneumonia(Sun et al.,combined tavailable ginvasive intants of N.identified twhich weretion to eigwere foundtified as eswere 16 sucurrently udidates.

Anotherin vitro expJohn Mekaogy was deare specifica bacterialgene that atauxotroph.growth in tgene, in whof the pathoing transcrsions weregenes. Thisan attenuatmay not bthe years, ttem, one oet al., 1995orescent prin vivo, dedifferentialFalkow, 19used to idenet al., 1996Y. enteroco(Camilli anal., 1998).

quire the knowledge of genome sequence for its appli-cation; however, the availability of genome sequencesdoes facilitate its use. Other complementary antigen

very alibrariwithnationy et alwith Suence

With td direach c

ly intrne red Salmnizat

ng ranwnstrnatingand chr prot

he clo

roteom

the ping onsis. Prill co

ion inin-seprometompo). ProThe fiheir pifferenin expin und

of pras ma

njuncturfaceor theordermonia1997), Salmonella typhimurium (Hensel etV. cholerae (Chiang and Mekalanos, 1998),terocolitica (Darwin and Miller, 1999), S.e (Polissi et al., 1998) and N. meningitidis2000). In case of N. meningitidis, Sun et al.he use of STM together with two publiclyenome sequences. Using an infant model offection, a library of 2850 insertional mu-meningitidis was scored and 73 genes werehat were essential for bacteraemia, many of

of unknown function. Moreover, in addi-ht known virulence genes, 65 novel genes, none of which had previously been iden-sential to infection in vivo. Also identifiedrface-expressed candidate antigens that arender investigation as potential vaccine can-

technology that uses gene expression isression technology (IVET), developed bylanos (Mahan et al., 1993). This technol-signed to identify promoters of genes thatally induced in host tissues. IVET requiresstrain carrying a mutation in a biosynthetictenuates growth in vivo, for example a purAThe biosynthetic function, essential for thehe host, is provided by a promoterless purAich fragments obtained from random librarygens chromosomal DNA supply the miss-

iption elements. The positively selected fu-then sequenced to identify in vivo-inducedIVET method necessitates the existence of

ing and complementable auxotrophy, whiche available in all microbial systems. Overhere have been variations to the IVET sys-f which is the use of an antibiotic (Mahan) or using the gene encoding the green flu-otein (gfp). The use of gfp to study genesvised by Valdivia and Falkow, is known asfluorescence induction, DFI (Valdivia and96, 1997a,b). IVET has successfully beentify virulence genes ofP. aeruginosa (Wanga,b), S. typhimurium (Mahan et al., 1995),litica (Young and Miller, 1997), V. choleraed Mekalanos, 1995) and S. aureus (Lowe etAs is the case for STM, IVET does not re-

discosionet al.vacci(Barret al.of seqtion.ligateapproinateimmusis animmuclonitor dovaccimidsconfetify t

6. P

Inassayanalyand wfunctprotespecttein c2001eas.

and tis dprotetionstudysuchin coand stant f

Inpneupproaches include whole genome expres-es (such as the study carried out by PelicicN. meningitidis (Pelicic et al., 2000), DNA

(Barry et al. with Mycoplasma pulmonis., 1995)) and genomic peptide libraries (Etz. aureus (Etz et al., 2002)). The availabilitydata has been exploited in DNA immuniza-

he genome data, ORFs can be amplified andctly into DNA immunization vectors. Thisould be useful for bacteria that are predom-acellular in the host and that elicit cellularsponses such as Chlamydia, M. tuberculo-onella. Barry et al. used expression library

ion with M. pulmonis. This method involvesdom fragments of bacterial DNA in a vec-eam of a promoter active in eukaryotic cells,animals with libraries of recombinant plas-allenging with Mycoplasma. Libraries that

ection can then be further analyzed to iden-nes responsible for protection.

ics

ast, protein analysis has been performed bye protein at a time, with very little paralleloteomics is the large-scale study of proteinsntribute greatly to the understanding of genethe post-genomic era. Recently, advances inaration technologies, combined with massry, have allowed the elucidation of total pro-nents of a given cellular population (Grandi,teomics can be divided into three main ar-rst is large-scale identification of proteinsost-translational modifications; the secondtial display proteomics for comparison ofression levels: this could have an implica-erstanding certain disease; the third is theoteinprotein interactions using techniquesss spectrometry. These three applicationsion with the characterization of membrane-associated proteins are particularly impor-vaccine development.to characterize the surface proteins of C.e, Montigiani et al. used the approach of ge-


nomics combined with proteomics (Montigiani et al.,2002). The authors purified 124 recombinant proteins,which were used to raise antibodies to test for theirability to bFACS bind53 putativespecificitytal proteinsseparated bjected to Wthen analyproaches alproteins suwhere protpathogenesStreptococcinuenzae

An attraof this techanalysis (Sscreen andvaccines ca

One exaS. aureusteins prepatwo-dimenmunoblottisera cominfrom S. aurlated and aidentificativaccine can

A similCell surfacrated usingsera from Hegy, the auteins, whicet al., 2002outer memidentified 6immunoreaet al., 2004

Ariel etanalysis inapplied toentire B. ain silico, u

putative vaccine candidates. This screening allowedthe identification of 520 open reading frames products,most of them putative surface exposed or exported

ins. Pprep

veralo test

sera

e comimmu

immuconc

omicsfor thee undnove

ase in

ompa

e sysdifferf contnew c

y of tsis ofnd ev

that oc; Schoetweemationorgane tec

ed to N). Themeninizes aensa

r specis, buto mafic gends. Diarrayngitidn withrent suind to the surface of chlamydial cells in aing assay. This led to the identification ofsurface-exposed proteins. To confirm the

of the sera used in the FACS analysis, to-from elementary bodies preparations were

y two-dimensional electrophoresis and sub-estern blot analysis. The positive spots werezed by mass spectrometry. All these ap-lowed the identification of surface-exposeditable for a novel vaccine. Other exampleseomics has also been used to study bacteriais and identify vaccine candidates includeus agalactiae (Hughes et al., 2002) and H.

(Langen et al., 2000; Thoren et al., 2002).ctive application of proteomics is the usenique in combination with the serological

ERPA, SERological Proteome Analysis), toselect new in vivo immunogens, valuable asndidates (Klade, 2002).mple is the work of Vytvytska et al., on(Vytvytska et al., 2002). A surface pro-ration from S. aureus was resolved by

sional electrophoresis and analyzed in im-ng using two pools, each consisting of fiveg from healthy donors or patients sufferingeus infections. Twenty-one spots were iso-nalyzed in mass spectrometry allowing theon of 15 proteins including known and noveldidates.

ar approach has been applied to H. pylori.e proteins of H. pylori were extracted, sepa-2D gels and analyzed in Western blot using. pylori-infected patients. Using this strat-

thors identified two new immunogenic pro-h may be used for vaccine development (Utt). Very recently, Baik et al. have isolated thebrane proteins of H. pylori strain 26695 and2 spots on 2D gels; nine of them resultedctive with sera from infected patients (Baik).

al. have used the serological proteomecombination with a bioinformatic approachB. anthracis (Ariel et al., 2003). First, thenthracis draft chromosome was screenedsing computational analyses, to identify

protebraneof seand tusingThesof 38vivo

Inprotetoolfor thfyingincre

7. C

Thfromcus o

lot ofabilitanalysals afiles2001son binformicro

Thappli2002ofN.coloncomm

eithecoccu

entlyspeciislanmicromeniactiodifferoteomic analysis of a B. anthracis mem-aration was used to verify the expressionmembrane-associated candidates selected

their immunoreactivity by immunoblotting,from B. anthracis-immunized animals.

bined approaches allowed the identificationnoreactive spots; eight of them resulted in

nogens.lusion, classical proteomics and immuno-approaches demonstrate to be a powerfulidentification of novel bacterial antigens,

erstanding of protein function and in identi-l vaccine components. Their use is likely tothe following years.

rative genomeproteome technologies

tematic comparison of genomic sequencesent microorganisms represents a central fo-emporary genome analysis and provides aoncepts in bacterial pathogenesis. The avail-he different genomes allows a comparativerelated bacteria, pathogens versus commen-en of bacteria with similar pathogenic pro-cupy different host niches (Claverie et al.,olnik, 2002a). Microarray-based compari-n two related genomes can provide valuable

about the diversity and evolution of theseisms.

hnique of comparative genomics has been. meningitidis by Nassif et al. (Perrin et al.,

y used DNA arrays to compare the genomegitidiswith those ofN. gonorrhoeae, whichdifferent host niche and N. lactamica, a

l of the nasopharynx. They identified genesfic for Meningococcus or shared with gono-t absent in N. lactamica. However, differ-ny other pathogens, these meningococcal-es are not organized in large chromosomal

fferently, Grifantini et al, compared, usings, the differential gene expression in N.is and in N. lactamica after bacterial inter-

human epithelial cells. They found that abset of genes was activated by hostcell


contact in pathogen and commensal species (Grifantiniet al., 2002a).

The technique of comparative genome hybridiza-tion (CGH)to compareof S. agalastudy consistrains reprthe genomedeterminedysis revealeare highly vcommon toto all straincapable oal., 2002).to compareof related BB. cereus (ative genomnon-pathogto the viruof potentia

A similfication ofM. bovis, bIn this casidentify gehad been lo(Cockle et

To analand commmade use omicroarraynon-pathogtraintestinathree pathosal and laban E. colprobes speExPEC, IPdistributioncommensa

Examplclude the sstrains of Mgens presential vaccin

Mattow et al., 2001). The authors identified 96 spotdifferences between the strains when they analyzedwhole-cell lysates. Fifty-six spots were found to be ex-

ve to Mass speeen puis studtes weDITpoteny of sossion

pproacproduously) and000).the la

e analgenicozzi,

002),r et a

., 200).

onclu

enomihes tg bah sta

me anmongover,ce infparad

is avgen cny prof theism.

omparhes, tentifi

sed prntly rehas recently been exploited by Tettelin et al.the complete gene repertoire of 22 strainsctiae (group B Streptococcus, GBS). Thested of the whole-genome hybridization ofesenting all nine known GBS serotypes with

of a serotype V isolate of which they hadthe complete genome sequence. The anal-d a number of regions of the genome thatariable and, more importantly, those genesall strains. The group of genes common

s contains the best candidates for a vaccinef serotype cross-protection (Tettelin etThe same technique has also been appliedthe genome of B. anthracis with 19 strainsacillus species, such as B. thuringensis andRead et al., 2003). Previously, a compar-e analysis among B. anthracis and other

enic Bacillus species had been restrictedlence plasmid pXO1 for the identificationl vaccine candidates (Ariel et al., 2002).ar concept has been applied to the identi-antigens conserved in M. tuberculosis andut absent from the Pasteur vaccine strain.e, bioinformatics tools have been used tones conserved in the two species, but whichst from the vaccine strain during evolutional., 2002).yze the genome plasticity in pathogenicensal E. coli isolates, Dobrindt et al.f a whole-genome approach. Using DNA

s, the presence of all translatable ORFs ofenic E. coli K-12 was investigated in 26 ex-l and intestinal pathogenic E. coli isolates,genicity island deletion mutants, commen-oratory strains. In addition, they developedi pathoarray, which consists of hundredscific for virulence-associated genes ofEC and Shigella, in order to evaluate theof these genes among the pathogenic and

l strains used (Dobrindt et al., 2003).es of comparative proteomics strategies in-tudy performed between M. tuberculosis. bovis BCG strains, with the idea that anti-t only in the virulent strain will be poten-

e candidates (Jungblut et al., 1999, 2000;

clusiby mhas bIn thisolaMALtheirabilitexprelar agensprevi2000al., 2

Inteompathoand Pal., 2Glaseet al2003

8. C

Gproacsectinproacgenofies aMorequennew

assaypathoout aedgeorgan

Cproacthe idexpodista. tuberculosis, of which 32 were identifiedctrometry. Furthermore, a very recent workblished on H. pylori (Govorun et al., 2003).y, proteome maps of four H. pylori clinicalre obtained using 2D-electrophoresis andOFmass-spectrometry. In order to evaluatetial as suitable vaccine candidates, the vari-me H. pylori proteins and the level of theirin the isolates have been evaluated. Simi-hes for the identification of putative anti-ced by this pathogen had been performedusing both DNA microarray (Salama et al.,comparative proteome analysis (Jungblut et

st years, the comparative genome and pro-ysis has been widely applied to many otherspecies such as S. pneumoniae (Oggioni2001), Streptococcus pyogenes (Smoot etL. monocytogenes (Buchrieser et al., 2003;l., 2001), Campylobacter jejuni (Pearson3) and Yersinia pestis (Hinchliffe et al.,

sions

cs has introduced a new paradigm in ap-o bacterial pathogenesis. Instead of dis-cterial components in vitro, the new ap-rts with the complete information on thed on the gene products and then identi-these the important factors in virulence.

the availability of complete genome se-ormation on many pathogens has led to aigm in vaccine development. If a suitableailable, every protein synthesized by thean be tested as a vaccine candidate with-ior selection based on incomplete knowl-

pathogenicity and immunogenicity of the

ed to conventional microbiological ap-he genome analysis of MenB has allowedcation of a higher number of novel surface-oteins, which are highly conserved amonglated strains and are also able to induce bac-


tericidal antibodies. These novel antigens hopefullywill be the basis for the clinical development of avaccine not only against group B Meningococcus, butalso againsNeisseriae.

Many intive vaccincomplete gand virusesdevelopmespective, wantigens fonear futurebacterial in

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Biotechnology and vaccines: application of functional genomics to Neisseria meningitidis and other bacterial pathogensIntroductionConventional vaccinology versus reverse vaccinologyReverse vaccinology applied to group B MeningococcusDNA microarray technologyInvestigating gene expression in vivoProteomicsComparative genome-proteome technologiesConclusionsReferences

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