Journal of Biotechnology 113 (2004) 1532

Biotechnology and vaccines: application of functional genomics to

Neisseria meningitidis and other bacterial pathogens
Davide Serruto, Jeannette Adu-Bobie, Barbara Capecchi,
Rino Rappuoli , Mariagrazia Pizza, Vega Masignani
IRIS, Chiron Vaccines,Via Fiorentina 1, 53100 Siena, Italy
Received 9 October 2003; received in revised form 9 March 2004; accepted 19 March 2004

Abstract
Since its introduction, vaccinology has been very effective in preventing infectious diseases. However, in several cases, the
conventional approach to identify protective antigens, based on biochemical, immunological and microbiological methods,
has failed to deliver successful vaccine candidates against major bacterial pathogens. The recent development of powerful
biotechnological tools applied to genome-based approaches has revolutionized vaccine development, biological research and
clinical diagnostics. The availability of a genome provides an inclusive virtual catalogue of all the potential antigens from which
it is possible to select the molecules that are likely to be more effective. Here, we describe the use of reverse vaccinology,
which has been successful in the identification of potential vaccines candidates against Neisseria meningitidis serogroup B
and review the use of functional genomics approaches as DNA microarrays, proteomics and comparative genome analysis for
the identification of virulence factors and novel vaccine candidates. In addition, we describe the potential of these powerful
technologies in understanding the pathogenesis of various bacteria.
2004 Elsevier B.V. All rights reserved.
Keywords: Vaccines; Genomics; Reverse vaccinology; Microarray; Proteomics

1. Introduction
Approaches to vaccine development have experienced remarkable progress during the last century.
Most of the vaccines currently available were gen Corresponding author. Tel.: +39-0577-243414;
fax: +39-0577-243564.
E-mail address: rino rappuoli@chiron.com (R. Rappuoli).

0168-1656/$ see front matter 2004 Elsevier B.V. All rights reserved.
doi:10.1016/j.jbiotec.2004.03.024

erated a long time ago and are based on killed or

live-attenuated microorganism, on toxins detoxified
by chemical treatment, on purified antigens and on
polysaccharide conjugated to proteins. The knowledge of the pathogenesis of many microorganisms, the
identification of the main virulence factors and the
characterization of the immune response after infection have been fundamental for the design of secondgeneration vaccines mainly based on highly purified

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antigenic components (Rappuoli and Del Giudice,

1999).
The development of prophylactic vaccines to prevent diseases has made an essential contribution to the
improvement of human health during the 20th century.
Devastating diseases such as smallpox and polio have
been totally or almost completely eradicated. However,
there are many infectious diseases still waiting for efficacious formulations and many emerging pathogens.
For these reasons, novel vaccines together with new
ways to discover and produce them are needed.
Recently, vaccine research and development has
gained new impetus for a number of reasons. First, it
is clear that our fight against bacterial infection is not
over; secondly, vaccines represent the most cost effective of all medical interventions (Rappuoli et al., 2002).
However, most importantly, the rapid development of
new technologies has allowed to overcome technological barriers that used to limit vaccine development.
The genomic revolution is one of the most important
technological advances.
The World Health Organization (WHO) recently released a report titled Genomics and World Health that
highlights the potential of genomics to improve global
health (WHO, 2002).
A pioneer study by Daar et al. identified the 10 most
promising biotechnologies for improving health in developing countries in the next 510 years (Daar et al.,
2002). In this study, one of the most highly rated categories was Recombinant technologies to develop vaccines against infectious diseases.
Modern recombinant DNA technology has been
used to produce subunit vaccines based on specific antigens. The first of these new vaccines was the highly
pure surface antigen of the hepatitis B virus (Andre,
1990). The second was the subunit vaccine against Bordetella pertussis containing three highly pure proteins.
The latter vaccine also pioneered the use of structure
function studies to produce genetically altered pertussis toxin that lacked toxicity but maintained an unaltered antigenic conformation (Pizza et al., 1989). These
novel approaches led to the development of a paradigm
for vaccine research, which has persisted for the last
two decades. In this approach, the microorganism is
studied from the point of view of pathogenicity and
immunology in order to identify factors involved in
virulence that may be suitable as vaccine candidates.
This methodology has been used for the development

of most new vaccines currently available or in clinical

development.
Among them, the recombinant vaccine against
Bacillus anthracis consists of the use of the protective
antigen PA, which corresponds to the beta subcomponent of the anthrax edema factor or lethal factor (EF or
LF), and which is recognized as the major immunogen
produced by the bacterium (Turnbull, 1991). Although
able to induce good protective immunity, vaccination
by PA-based vaccines requires multiple immunizations, underlying the need to develop more efficacious
vaccines or alternative vaccination regimens.
Several recombinant-based approaches are also being carried out for the development of new cholera vaccines. An example is represented by the commercially
available oral vaccines, such as the whole cell combined with a non-toxic form of the B subunit (WCBS)
and the live-attenuated Vibrio cholerae strain, produced
with recombinant techniques and not expressing the enzymatically active subunit of the cholera toxin (CtxA).
This vaccine is safe, immunogenic and generally well
tolerated; however, the protective effect is not long lasting and repeated booster doses are recommended (Ryan
and Calderwood, 2000). Additional live-attenuated V.
cholerae vaccines are based upon the removal of the
enzymatically active subunit of cholera toxin or of the
entire cholera toxin genetic element by molecular genetic techniques and are in various stages of analysis.
Another interesting example of a single antigenbased vaccine is that against Borrelia burgdorferi, the
etiologic agent of lyme borreliosis. In this case, the
outer surface lipoprotein OspA has been shown to possess protective activity against the disease (Steere et
al., 1998). An OspA vaccine, LYMErix, which was developed by SmithKline Beecham, has been recently
approved for human use; however, a major deficiency
of OspA-based vaccines is the variability of the antigen
among the various species of B. burgdorferi (Wilske et
al., 1996). For this reason, alternative approaches taking into account the use of single conserved portions
of the protein are currently being pursued (Luft et al.,
2002).
Furthermore, protein-based approaches are also
being evaluated for other important human pathogens,
such as Moraxella catarrhalis and non-typeable
Haemophilus inuenzae (McMichael and Green,
2003), Streptococcus pneumoniae (Swiatlo and Ware,
2003) and for some viruses, such as hepatitis C (HCV)

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

(Hsu et al., 1999), human immunodeficiency virus

(HIV) (Amara and Robinson, 2002), human papilloma
virus (Rohan et al., 2003) and EpsteinBarr virus
(Finerty et al., 1994).
Finally, it is worth mentioning the use of live viral and bacterial vectors as delivery systems for recombinant vaccines (Stephenson, 2001; Medina and
Guzman, 2001). On the basis of genetic information, it
is now possible to select a priori the bacterial and viral
genes to inactivate to improve the safety profile of live
vectors and to identify the potential antigens to use as
immunogens. The major advantage in using live vectors is that the heterologous antigen is presented to the
immune system by the host in its native conformation
and to the right compartment.
Other two most highly rated categories presented in
the study by Daar et al. were Sequencing pathogen
genomes to understand their biology and to identify
new antimicrobials and Bioinformatics to identify
drug targets and to examine pathogenhost interactions.
Automated DNA sequence analysis, together with
the availability of new bioinformatic tools, has revolutionized the field of biology and medicine, opening the new era of genomic science (Andrade and
Sander, 1997; Brutlag, 1998; Fraser et al., 2000). The
complete genome sequence of a bacterium can be
obtained in a brief period of time using the shotgun sequencing strategy. This technique was successfully used at The Institute for Genomic Research
(TIGR) in 1995, to determine the genome sequence
of H. inuenzae (Fleischmann et al., 1995). In the
last few years, the number of available genomes has
grown considerably (Fig. 1). Today, a quick look at the
genome databases reveals the level of knowledge on
the bacterial world: in addition to the 156 completed
genome sequences, more than 400 other microorganisms are being sequenced in various laboratories
around the world (GOLD Genomes OnLine database
at http://wit.integratedgenomics.com/GOLD/). This
panel of bacterial genomes already covers most of
the pathogens impacting heavily on human health and
therefore of interest for vaccine researchers.
Bioinformatics is essential to interpret the immense
amount of information contained in whole genome sequences (genomic mining). A variety of software can
be used to assign gene functions and predict key features such as topology, molecular weight, pI and sol-

17

ubility. Moreover, a putative function can be assigned

to each open reading frame (ORF) on the basis of a
homology to known proteins. Sophisticated computer
programs are also available to predict cellular localization of newly identified ORFs, so that it becomes possible to choose potentially surface-exposed proteins.
Complementary to in silico antigen discovery approaches are strategies referred to as Functional Genomics. These approaches include the large-scale
analysis of gene transcription, using DNA microarray
technology, the whole set of proteins encoded by an organism (proteomics) using two-dimensional gel electrophoresis and mass spectrometry and the comparative
genomeproteome technologies.

2. Conventional vaccinology versus reverse

vaccinology
The conventional approaches to produce vaccines
are based on the cultivation of the microorganism in
laboratory conditions from which single components
are isolated individually by using biochemical and microbiological methods. Each antigen is produced in
pure form and finally tested for its ability to induce
an immune response. However, although successful in
many cases, this approach presents several limitations.
It needs to grow the pathogen in vitro, so it is not applicable to non-cultivable microorganisms, and in many
cases, the antigens expressed during infection are not
produced in laboratory conditions. This method can
employ many years to identify a protective and useful
antigen and has failed to provide a vaccine against those
pathogens that did not have obvious immunodominant
protective antigens (i.e. capsule or toxins).
The reverse approach to vaccine development takes
advantage of the genome sequence of the pathogen. The
genome represents, virtually, a list of all the proteins
that the pathogen can express at any time. It becomes
possible to choose potentially surface-exposed proteins
in a reverse manner, starting from the genome rather
than from the microorganism. In the field of vaccinology, genomics represent a fantastic reservoir of genes
that can be screened and tested as vaccine candidates.
This novel approach has been coined reverse vaccinology (Fig. 2) (Rappuoli, 2000, 2001).
Here, we describe how a reverse vaccinology approach has been successful to identify novel potential

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

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

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

19

Fig. 2. Comparison of methodologies and time between conventional and reverse vaccinology to vaccine development.

vaccine candidates against the human pathogen Neisseria meningitidis serogroup B and illustrate the application of functional genomics to vaccine research.

3. Reverse vaccinology applied to group B

Meningococcus
N. meningitidis is the major cause of meningitis and
sepsis, two devastating diseases that can kill children
and young adults. Meningococcus is a Gram-negative
bacterium that colonizes asymptomatically the upper
nasopharynx tract of about 515% of the human
population. However, for reasons yet unknown, in a
significant number of cases, the bacterium can traverse
the epithelium and reach the bloodstream causing
septicemia. From the blood, Meningococcus is able to
cross the blood brain barrier and infect the meninges,
causing meningitis.
The incidence of meningococcal disease ranges
from 13 to 1025/100,000 cases in industrialized
or developing countries, respectively, and despite se-

vere antibiotic therapies, the mortality rates remain

high.
N. meningitidis can be classified in 13 serogroups on
the basis of their polysaccharides capsule. However,
more than 95% of total cases of invasive disease are
caused by five major serogroups: A, B, C, Y and W135.
Efficacious vaccines composed from purified capsule
polysaccharides of serogroups A, C, Y and W135 are
currently available. More recently, a new generation of
glycoconjugate vaccine against serogroup C has been
licensed. This vaccine, tested in UK, has shown an efficacy of 97% in adolescents and 92% in toddlers, preventing more than 500 cases in one year (Ramsay et
al., 2001). A similar approach will be extended to the
other serogroups A, Y and W135.
The critical point remains with serogroup B (MenB)
for which the polysaccharide-based vaccine approach
cannot be used. In fact, the major component of the
MenB capsule is the (28)-linked N-acetylneuraminic
acid, a common carbohydrate present also in the mammalian tissue. Therefore, this polysaccharide, being a
self-antigen, is poorly immunogenic and may elicit au-

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

toimmunity. An alternative approach to vaccine development is based on surface-exposed proteins contained

in the outer membrane vesicles (OMVs). However, due
to the high sequence and antigenic variability of the
antigens present in the OMVs, these vaccines have
shown to elicit protective antibodies but only against
the homologous strain (Bjune et al., 1991; Tappero et
al., 1999; de Moraes et al., 1992).
Forty years of studies based on classical approaches
to vaccine research have failed to provide an efficacious
vaccine against MenB that remains one of the principle
causes for bacterial meningitis in industrialized countries. To overcome the latter obstacle, the new approach
named reverse vaccinology was applied to MenB
(Pizza et al., 2000). To this end, the complete genome of
the virulent strain MC58 was sequenced in collaboration with TIGR using the shotgun strategy. The MenB
genome consists of 2,272,352 base pairs with an average of G + C content of 51.5%. The 83% of the genome
codes for 2158 ORFs. Out of these, 1158 have a putative
biological role assigned on the basis of their similarity
with known proteins, whereas the remaining 1000 have
not a predicted function (Tettelin et al., 2000).
Based on the concept that surface-exposed antigens are more susceptible to antibodies recognition and
therefore are the most suitable candidates for a vaccine, the full genome was screened using bioinformatics tools in order to select open reading frames coding
for putative surface-exposed or secreted proteins.
All the putative ORFs were analyzed using computer programs as PSORT or SignalP, to predict the
signal peptide sequences, TMPRED to identify the putative hydrophobic membrane regions and MOTIFS to
identify lipoproteins. Finally, ORFs coding for proteins with homology to known virulence factors of
other bacteria were also included. This screening allowed the identification of 600 putative ORFs that were
classified on the basis of their predicted features: secreted or outer membrane proteins (13%), lipoproteins
(20%), periplasmic proteins (27%), inner membrane
proteins (34%) and proteins with interesting homology (6%). All these ORFs were amplified by PCR and
cloned in Escherichia coli, in order to express them
as N-terminal glutathione-S-transferase (GST) or Cterminal histidine-tag fusion. Three hundred and fifty
recombinant proteins were expressed, purified and used
to immunize mice. The sera obtained were then tested
with several assays. First of all, sera were analyzed

in Western blot on total cell lysate and outer membrane preparation to confirm that each protein was really expressed in vivo and localized in the outer membrane. ELISA and FACS analysis on whole-cell bacteria were performed to verify the surface-localization
of the expressed proteins. Finally, all the sera were
tested for their complement-mediated bactericidal activity, which is known to correlate with the protection
in humans. From this screening, 91 proteins resulted
positive in at least one assay, and out of them, 28 were
positive in the bactericidal assay (Fig. 3). Among these,
few candidates were selected and subjected to further
studies.
As mentioned before, one of the main problems to
face in the design of a vaccine against MenB is the
sequence variability of the antigens among different
strains. For example, the most abundant antigen of
MenB, PorA, is extremely variable and able to confer
protection only against the homologous strain. In view
of that, the nine best vaccine candidates selected by the
reverse vaccinology approach were analyzed for their
sequence variability using a representative panel of 31
strains, inclusive of N. meningitidis, N. cinerea, N. lactamica and N. gonorrhoeae. Each gene was amplified
by PCR from all the 31 selected strains and sequenced.
The sequences were subjected to multiple alignments
to verify the level of homology among the different
alleles. Surprisingly, hypervirulent regions were identified only in the case of two antigens, whereas all the
other proteins were highly conserved, with percentage
of identity of about 99%. Finally, these conserved antigens were able to induce complement-mediated bacterial killing in a subset of strains for which a suitable
complement source was available. Reverse vaccinology allowed the identification in a few years of many
antigens that now could be considered as basis for developing a vaccine against MenB.
Interestingly, the availability of the entire genome
provides an inexhaustible source of unknown and undescribed proteins, several of them sharing attractive
homologies with known virulence factors of other bacteria. To verify whether they could have a role also
in the pathogenesis of Meningococcus, some of these
proteins have been further characterized from the biochemical and functional point of view. A brief description of them is reported in Table 1.
In the last few years, several groups have followed
the path of pioneer work on MenB, utilizing the ap-

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

21

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 the
antigens are expressed and surface-exposed; the bactericidal assay is used to evaluate the complement-mediated bacterial killing activity of
antibodies.

Table 1
Description of some of the novel MenB antigens identified
N. meningitidis antigen

Description

Reference

GNA33

Membrane-bound lytic transglycosylase (MltA) of N. meningitidis

GNA992
NadA
GNA1870
App
NarE

A putative adhesin homologue to Hsf and Hia proteins

A new adhesin vaccine candidate
Surface-exposed lipoprotein as vaccine candidate
An autotransporter adhesin with autocatalytic serine protease activity
A novel ADP-ribosylating enzyme

Granoff et al., 2001; Jennings et al., 2002;

Adu-Bobie et al., 2004
Scarselli et al., 2001
Comanducci et al., 2002
Masignani et al., 2003b
Serruto et al., 2003
Masignani et al., 2003a

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

proach of reverse vaccinology and functional genomics

(microarray, proteomics and comparative analysis) to
identify new vaccine candidates. Bacteria that have
been studied include several human pathogens such as
B. anthracis, Chlamydia pneumoniae, S. pneumonia,
Staphylococcus aureus, Porphyromonas gingivalis,
Listeria monocytogenes and Mycobacterium tuberculosis. A comprehensive list of the different genomic approaches described in this review is reported in Table 2.

4. DNA microarray technology

DNA microarray (or microchips) is a recently developed genomic technology and is one of the most
powerful tools for the study of the transcriptome, the
complete set of transcripts of an organism (Brown and
Botstein, 1999; Cheung et al., 1999; Lipshutz et al.,
1999; Lockhart and Winzeler, 2000). Recent advances
in the technology of massive parallel gene expression profiling using microarrays are revolutionizing
biological research, clinical diagnostics and vaccine
discovery.
Microarray technology can be used for several
applications including gene expression profiling,
genotyping and DNA sequencing. As a result, gene
expression array data can be analyzed on at least three
levels. The first level is that of single genes; the aim
is to establish whether each isolated gene behaves
differently in different conditions. The second level is
that of multiple genes, where clusters of genes are analyzed in terms of common functionalities, interactions
and co-regulation. Finally, the third level is that of
cellbiology metabolism pathways, where the aim is to
understand the underlying gene and protein networks
that are responsible for the various patterns (Hatfield
et al., 2003). Researchers are using this technology
to identify genes that are differentially expressed in
response to alteration in environmental parameters and
to evaluate mutations in regulatory and metabolic pathways. Moreover, another purpose is to capture the transcriptome of bacteria growing within infected cells and
tissues and, as a result, to disclose the host-adapted transcriptional reaction. The applications of DNA microarray technology in all these biological fields have been
extensively described elsewhere by several excellent
reviews (Schoolnik, 2002b; Cummings and Relman,
2000; Kato-Maeda et al., 2001; Conway and School-

nik, 2003). In this section, we describe some of the

recent studies that provide forceful proof that the
microarray technology allows to examine the dynamics of a whole biological system by simultaneously
interrogating the expression of thousands of genes.
Using S. aureus GeneChip, Dunman et al. have identified genes that are regulated by agr and/or SarA, the
two regulators of the microorganisms virulence response (Dunman et al., 2001). Mekalanos et al. demonstrated that quorum-sensing regulators are involved in
V. cholerae virulence. Gene arrays were used to profile
transcription in the wild-type strain and in the luxO mutant and, consequently, to define the LuxO regulon (Zhu
et al., 2002). In a recent study, Falkow et al. investigated
the whole-genome expression profiling of Helicobacter pylori grown in vitro. This time course analysis
illustrates a major switch in gene expression at the late
log phase-to-stationary phase transition (Thompson et
al., 2003). The iron-activated and -repressed genes of N.
MenB were identified by transcriptome analysis: using
DNA microarray, computational and in vitro studies,
Grifantini et al. defined the Fur regulon of this human
pathogen (Grifantini et al., 2003).
The complex interaction between host and pathogen
is also being explored using microarrays. Virulence
gene expression can be monitored by growing the
pathogens in the appropriate in vivo models (cell cultures and/or animals) and, after recovering the bacteria
for RNA preparation and labelling, the gene activity
is analyzed and compared with the expression of the
genes under in vitro conditions.
There are several studies focused on hostpathogen
interactions using DNA microarrays and they include
the interactions between intestinal epithelial cells and
Salmonella (Eckmann et al., 2000), human promyelocytic cells and L. monocytogenes (Cohen et al., 2000),
bronchial epithelial cells and B. pertussis (Belcher et
al., 2000), epithelial cells and Pseudomonas aeruginosa (Ichikawa et al., 2000), mouse gastric epithelial
cells with and without exposure to H. pylori (Mills et
al., 2001), HeLa cells infected with Chlamydia trachomatis (Xia et al., 2003), human macrophage response to infection with M. tuberculosis (Wang et al.,
2003), global response of human intestinal epithelial
cells to Shigella exneri invasion (Pedron et al., 2003).
However, these studies consider gene activation
from the host perspective. Recently, the DNA microarray technology has been applied to study the

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

23

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

Approaches

References

Bacillus anthracis

Reverse vaccinology
Comparative genome analysis
Serological proteome analysis

Ariel et al., 2002

Read et al., 2003
Ariel et al., 2003

Bordetella pertussis
Campylobacter jejuni
Chlamydia pneumoniae

Microarray
Comparative genome analysis
Proteomics/reverse vaccinology

Belcher et al., 2000

Pearson et al., 2003
Montigiani et al., 2002

Chlamydia trachomatis

Microarray
Microarray

Xia et al., 2003

Belland et al., 2003

Escherichia coli
Haemophilus inuenzae
Helicobacter felis

Comparative genome analysis

Proteomics
Microarray

Dobrindt et al., 2003

Langen et al., 2000; Thoren et al., 2002
Mueller et al., 2003

Helicobacter pylori

Microarray
Microarray
Comparative genome analysis
Comparative proteome analysis
Serological proteome analysis

Mills et al., 2001

Thompson et al., 2003
Salama et al., 2000
Jungblut et al., 2000; Govorun et al. 2003
Utt et al., 2002; Baik et al., 2004

Listeria monocytogenes

Microarray
Comparative genome analysis

Cohen et al., 2000

Glaser et al., 2001; Buchrieser et al., 2003

Mycobacterium tuberculosis

Microarray
Microarray
STM
Comparative genome analysis
Comparative proteome analysis

Wang et al., 2003

Schnappinger et al., 2003; Wilson et al., 1999
Camacho et al., 1999
Cockle et al., 2002
Jungblut et al., 1999; Mattow et al., 2001

Mycoplasma pulmonis

DNA vaccination

Barry et al., 1995

Neisseria meningitidis

Reverse vaccinology
Microarray
STM
Whole genome expression library
Comparative genome analysis

Pizza et al., 2000

Grifantini et al., 2002a,b, 2003; Kurz et al., 2003
Sun et al., 2000
Pelicic et al., 2000
Perrin et al., 2002

Porphyromonas gingivalis

Reverse vaccinology

Ross et al., 2001

Pseudomonas aeruginosa

Microarray
IVET

Ichikawa et al., 2000

Wang et al., 1996a,b

Salmonella typhimurium

Microarray
STM
IVET
DFI

Eckmann et al., 2000

Hensel et al., 1995
Mahan et al., 1995
Valdivia and Falkow, 1996, 1997a

Shigella exneri

Microarray

Pedron et al., 2003

Staphylococcus aureus

Microarray
STM
IVET
Genomic peptide libraries
Serological proteome analysis

Dunman et al., 2001

Coulter et al., 1998; Mei et al., 1997
Lowe et al., 1998
Etz et al., 2002
Vytvytska et al., 2002

Streptococcus agalactiae

Comparative genome analysis

Proteomics

Tettelin et al., 2002

Hughes et al., 2002

Streptococcus pneumoniae

Reverse vaccinology
STM

Wizemann et al., 2001

Polissi et al., 1998

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

Table 2 (Continued )
Bacterium

Approaches

References

Comparative genome analysis

Oggioni and Pozzi, 2001

Streptococcus pyogenes

Comparative genome analysis

Smoot et al., 2002

Vibrio cholerae

Microarray
STM
IVET

Zhu et al., 2002

Chiang and Mekalanos, 1998
Camilli and Mekalanos, 1995

Yersinia enterocolitica

STM
IVET

Darwin and Miller, 1999

Young and Miller, 1997

Yersinia pestis

Comparative genome analysis

Hinchliffe et al., 2003

Note: microarray studies indicated with an asterisk ( ) consider gene activation from the host perspective; all the other papers analyzed the
bacterial gene expression profile.

gene expression profile of human pathogens during

different stages of infection.
Belland et al. have characterized the genomic transcriptional profiling of the developmental cycle of C.
trachomatis. In this work, microarrays were used to
analyze the temporal expression of chlamydial genes
throughout the life cycle. This approach has provided
new findings about chlamydial gene products that control the differentiation stages and determine the nature
of the hostpathogen interaction. These genes encode
products that are chlamydial-specific and in certain
cases, have phylogenetic signatures that suggest eukaryotic origin. The authors hypothesize that Chlamydiae may have acquired these genes from the host and
have used them functionally to define their distinctive
biology. This work demonstrated also that microarray
technology is a powerful and indispensable tool to shed
light into the complex interactions between host and
chlamydiae because there are not genetic exchange
tools for the bacteria; consequently, many of the differently expressed genes would likely remain undiscovered without the application of microarray analysis
(Belland et al., 2003).
Another particularly informative study defined the
transcriptional adaptation of M. tuberculosis within
macrophages (Schnappinger et al., 2003). M. tuberculosis is able at adapting to long-term residence in
macrophage phagosomes because it blocks maturation
of phagosomes into phagolysosomes by controlling the
development of these compartments. Schoolnik and
collaborators captured the global expression profile of
this pathogen and identified the genes that are differently expressed by intraphagosomal M. tuberculosis in nave and INF--activated macrophages, com-

pared with bacteria grown in standard broth culture.

The study demonstrated that the bacterium modifies
the transcriptome in order to adapt to the phagosomal
environment by the induction of fatty acid-degrading
enzymes and DNA repair proteins and the production
of secreted siderophores to facilitate the acquisition of
iron. Finally, the authors used expression profiles of M.
tuberculosis exposed to various in vitro conditions of
growth to isolate situations that mimic the phagosomal
environment (Schnappinger et al., 2003).
Microarrays provide a powerful tool, not only to understand the regulation of gene expression in bacteria,
but also to discover new virulence genes, vaccine candidates and drug targets.
One example where microarray technology has been
successful to identify potential vaccine candidates, as
well as new virulence genes, is in the case of N.
meningitidis. In order to understand the pathology of
Meningococcus and for the identification of new vaccine candidates, DNA microarray technology has been
used to study gene regulation after interaction of N.
meningitidis to human epithelial cells (Grifantini et al.,
2002b). RNAs were isolated from adherent and nonadherent bacteria and comparatively analyzed on DNA
microarrays carrying the entire collection of PCRamplified MenB genes. The authors found that bacterial adhesion to epithelial cells altered the expression of approximately 350 genes: 189 genes were upregulated and 151 genes were downregulated while
seven were either upregulated or downregulated depending on the time point of infection. Most of
the regulated genes can be grouped into five major categories: adhesion genes, hostpathogen crosstalk genes, amino acids and selenocysteine biosyn-

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

thesis genes, DNA metabolism genes and hypothetical genes. Moreover, of the 12 adhesion-induced
surface-exposed antigens identified, five were able
to induce bactericidal antibodies. In conclusion, this
study shows that DNA microarray technology is able
to identify potential vaccine candidates and complement other genome mining methods such as reverse
vaccinology.
In an independent study, the transcriptional changes
of N. meningitidis were investigated in a model system
of three key steps of meningococcal infection. RNA
was isolated from Meningococci incubated in human
serum as well as adherent to human epithelial and
endothelial cells. The authors discovered that a wide
range of surface proteins which are induced under in
vivo conditions. These antigens could represent novel
candidates for a protein-based vaccine for meningococcal diseases (Kurz et al., 2003).
The first applications of DNA microarray in parasitology are in place (Rathod et al., 2002). The complete genome sequence of Plasmodium falciparum was
recently published and systematic approaches to the development of vaccines based on the completed genome
sequence are already in planning stages (Gardner et al.,
2002; Long and Hoffman, 2002). One approach is to
catalogue the expression of proteins at each stage of the
parasitic lifecycle using this technology and screening
the upregulated proteins using sera from immune subjects. Selected genes can then be cloned, expressed and
evaluated as vaccine components in challenge studies.
Recently, a pioneer work showed how microarray
expression profiling could be used to discover new
drugs and their mode of action. Wilson et al. used a
DNA microarray containing 97% of the ORFs of the
M. tuberculosis genome to examine changes in the gene
expression in response to the antituberculous drug isoniazid. They reported that isoniazid treatment of midlog phase bacterial cultures induced several genes that
encode proteins physiologically relevant to the drugs
mode of action. Other genes were induced and likely
mediate processes that are linked to the toxic consequences of the drug (Wilson et al., 1999). This study
points up how gene expression analysis can contribute
to the drug discovery process: exposure of a microorganism to a drug or compound of unknown mode of
action should elicit an expression profile that incriminates the affected pathway and even the target in the
pathway (Schoolnik, 2002b).

25

The understanding of the protective mechanism mediated by each vaccine is an essential prerequisite to design new rationally based vaccines. In a recent work,
Falkow and collaborators used gene expression profiling and immunohistochemical analysis to unravel the
mechanism of protection of a whole-cell sonicate vaccine of Helicobacter felis in mice (Mueller et al., 2003).
This approach, applied to other immunization strategies, will help to better understand the mechanism of
protection of several vaccine formulations.
In recent years, there have been enormous advances in DNA microarray technology and a remarkable amount of literature supporting its central role in
gene discovery, vaccine and drug development. However, because the results of pathogen gene expression
are influenced by the model system used, such results
must be interpreted cautiously. In addition, expression
data have limitations because mRNA levels may not reflect protein levels, and expression of a protein may not
always have a pathological consequence (Gygi et al.,
1999). Consequently, traditional biological, pathology
and toxicity studies remain necessary.

5. Investigating gene expression in vivo

Gene expression in vivo has been exploited in order to identify virulence genes, which is a key step in
vaccine design. A variety of methods have been formulated to isolate genes that are specially induced during an infection. One of the recent technologies used
in vaccine design, which does not strictly depend on
but is facilitated by genome sequencing, is the signature tagged mutagenesis (STM) developed by David
Holden (Hensel et al., 1995). A bacterial pathogen is
subjected to random transposon-mediated mutagenesis in such a way that each mutant is tagged with a
specific short DNA sequence tag. Comparison, by hydridization, of the tags found in arrays representing all
mutants capable of growing in vitro with mutants that
survive passage through an animal host identifies genes
essential for the infectious process. The advantage of
using this approach is that the technique allows for the
identification of attenuated mutants that fail to cause a
productive infection and therefore may be used as live
vaccines. Moreover, proteins identified as being essential for infection or disease are likely to be good candidates for inclusion in subunit vaccines. STM has been

26

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

successfully used to discover virulence genes from a

variety of bacterial species including M. tuberculosis
(Camacho et al., 1999), S. aureus (Coulter et al., 1998;
Mei et al., 1997), Salmonella typhimurium (Hensel et
al., 1995), V. cholerae (Chiang and Mekalanos, 1998),
Yersinia enterocolitica (Darwin and Miller, 1999), S.
pneumoniae (Polissi et al., 1998) and N. meningitidis
(Sun et al., 2000). In case of N. meningitidis, Sun et al.
combined the use of STM together with two publicly
available genome sequences. Using an infant model of
invasive infection, a library of 2850 insertional mutants of N. meningitidis was scored and 73 genes were
identified that were essential for bacteraemia, many of
which were of unknown function. Moreover, in addition to eight known virulence genes, 65 novel genes
were found, none of which had previously been identified as essential to infection in vivo. Also identified
were 16 surface-expressed candidate antigens that are
currently under investigation as potential vaccine candidates.
Another technology that uses gene expression is
in vitro expression technology (IVET), developed by
John Mekalanos (Mahan et al., 1993). This technology was designed to identify promoters of genes that
are specifically induced in host tissues. IVET requires
a bacterial strain carrying a mutation in a biosynthetic
gene that attenuates growth in vivo, for example a purA
auxotroph. The biosynthetic function, essential for the
growth in the host, is provided by a promoterless purA
gene, in which fragments obtained from random library
of the pathogens chromosomal DNA supply the missing transcription elements. The positively selected fusions were then sequenced to identify in vivo-induced
genes. This IVET method necessitates the existence of
an attenuating and complementable auxotrophy, which
may not be available in all microbial systems. Over
the years, there have been variations to the IVET system, one of which is the use of an antibiotic (Mahan
et al., 1995) or using the gene encoding the green fluorescent protein (gfp). The use of gfp to study genes
in vivo, devised by Valdivia and Falkow, is known as
differential fluorescence induction, DFI (Valdivia and
Falkow, 1996, 1997a,b). IVET has successfully been
used to identify virulence genes of P. aeruginosa (Wang
et al., 1996a,b), S. typhimurium (Mahan et al., 1995),
Y. enterocolitica (Young and Miller, 1997), V. cholerae
(Camilli and Mekalanos, 1995) and S. aureus (Lowe et
al., 1998). As is the case for STM, IVET does not re-

quire the knowledge of genome sequence for its application; however, the availability of genome sequences
does facilitate its use. Other complementary antigen
discovery approaches include whole genome expression libraries (such as the study carried out by Pelicic
et al. with N. meningitidis (Pelicic et al., 2000), DNA
vaccination (Barry et al. with Mycoplasma pulmonis
(Barry et al., 1995)) and genomic peptide libraries (Etz
et al. with S. aureus (Etz et al., 2002)). The availability
of sequence data has been exploited in DNA immunization. With the genome data, ORFs can be amplified and
ligated directly into DNA immunization vectors. This
approach could be useful for bacteria that are predominately intracellular in the host and that elicit cellular
immune responses such as Chlamydia, M. tuberculosis and Salmonella. Barry et al. used expression library
immunization with M. pulmonis. This method involves
cloning random fragments of bacterial DNA in a vector downstream of a promoter active in eukaryotic cells,
vaccinating animals with libraries of recombinant plasmids and challenging with Mycoplasma. Libraries that
confer protection can then be further analyzed to identify the clones responsible for protection.

6. Proteomics
In the past, protein analysis has been performed by
assaying one protein at a time, with very little parallel
analysis. Proteomics is the large-scale study of proteins
and will contribute greatly to the understanding of gene
function in the post-genomic era. Recently, advances in
protein-separation technologies, combined with mass
spectrometry, have allowed the elucidation of total protein components of a given cellular population (Grandi,
2001). Proteomics can be divided into three main areas. The first is large-scale identification of proteins
and their post-translational modifications; the second
is differential display proteomics for comparison of
protein expression levels: this could have an implication in understanding certain disease; the third is the
study of proteinprotein interactions using techniques
such as mass spectrometry. These three applications
in conjunction with the characterization of membrane
and surface-associated proteins are particularly important for the vaccine development.
In order to characterize the surface proteins of C.
pneumoniae, Montigiani et al. used the approach of ge-

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

nomics combined with proteomics (Montigiani et al.,

2002). The authors purified 124 recombinant proteins,
which were used to raise antibodies to test for their
ability to bind to the surface of chlamydial cells in a
FACS binding assay. This led to the identification of
53 putative surface-exposed proteins. To confirm the
specificity of the sera used in the FACS analysis, total proteins from elementary bodies preparations were
separated by two-dimensional electrophoresis and subjected to Western blot analysis. The positive spots were
then analyzed by mass spectrometry. All these approaches allowed the identification of surface-exposed
proteins suitable for a novel vaccine. Other examples
where proteomics has also been used to study bacteria
pathogenesis and identify vaccine candidates include
Streptococcus agalactiae (Hughes et al., 2002) and H.
inuenzae (Langen et al., 2000; Thoren et al., 2002).
An attractive application of proteomics is the use
of this technique in combination with the serological
analysis (SERPA, SERological Proteome Analysis), to
screen and select new in vivo immunogens, valuable as
vaccines candidates (Klade, 2002).
One example is the work of Vytvytska et al., on
S. aureus (Vytvytska et al., 2002). A surface proteins preparation from S. aureus was resolved by
two-dimensional electrophoresis and analyzed in immunoblotting using two pools, each consisting of five
sera coming from healthy donors or patients suffering
from S. aureus infections. Twenty-one spots were isolated and analyzed in mass spectrometry allowing the
identification of 15 proteins including known and novel
vaccine candidates.
A similar approach has been applied to H. pylori.
Cell surface proteins of H. pylori were extracted, separated using 2D gels and analyzed in Western blot using
sera from H. pylori-infected patients. Using this strategy, the authors identified two new immunogenic proteins, which may be used for vaccine development (Utt
et al., 2002). Very recently, Baik et al. have isolated the
outer membrane proteins of H. pylori strain 26695 and
identified 62 spots on 2D gels; nine of them resulted
immunoreactive with sera from infected patients (Baik
et al., 2004).
Ariel et al. have used the serological proteome
analysis in combination with a bioinformatic approach
applied to B. anthracis (Ariel et al., 2003). First, the
entire B. anthracis draft chromosome was screened
in silico, using computational analyses, to identify

27

putative vaccine candidates. This screening allowed

the identification of 520 open reading frames products,
most of them putative surface exposed or exported
proteins. Proteomic analysis of a B. anthracis membrane preparation was used to verify the expression
of several membrane-associated candidates selected
and to test their immunoreactivity by immunoblotting,
using sera from B. anthracis-immunized animals.
These combined approaches allowed the identification
of 38 immunoreactive spots; eight of them resulted in
vivo immunogens.
In conclusion, classical proteomics and immunoproteomics approaches demonstrate to be a powerful
tool for the identification of novel bacterial antigens,
for the understanding of protein function and in identifying novel vaccine components. Their use is likely to
increase in the following years.

7. Comparative genomeproteome technologies

The systematic comparison of genomic sequences
from different microorganisms represents a central focus of contemporary genome analysis and provides a
lot of new concepts in bacterial pathogenesis. The availability of the different genomes allows a comparative
analysis of related bacteria, pathogens versus commensals and even of bacteria with similar pathogenic profiles that occupy different host niches (Claverie et al.,
2001; Schoolnik, 2002a). Microarray-based comparison between two related genomes can provide valuable
information about the diversity and evolution of these
microorganisms.
The technique of comparative genomics has been
applied to N. meningitidis by Nassif et al. (Perrin et al.,
2002). They used DNA arrays to compare the genome
of N. meningitidis with those of N. gonorrhoeae, which
colonizes a different host niche and N. lactamica, a
commensal of the nasopharynx. They identified genes
either specific for Meningococcus or shared with gonococcus, but absent in N. lactamica. However, differently to many other pathogens, these meningococcalspecific genes are not organized in large chromosomal
islands. Differently, Grifantini et al, compared, using
microarrays, the differential gene expression in N.
meningitidis and in N. lactamica after bacterial interaction with human epithelial cells. They found that a
different subset of genes was activated by hostcell

28

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

contact in pathogen and commensal species (Grifantini

et al., 2002a).
The technique of comparative genome hybridization (CGH) has recently been exploited by Tettelin et al.
to compare the complete gene repertoire of 22 strains
of S. agalactiae (group B Streptococcus, GBS). The
study consisted of the whole-genome hybridization of
strains representing all nine known GBS serotypes with
the genome of a serotype V isolate of which they had
determined the complete genome sequence. The analysis revealed a number of regions of the genome that
are highly variable and, more importantly, those genes
common to all strains. The group of genes common
to all strains contains the best candidates for a vaccine
capable of serotype cross-protection (Tettelin et
al., 2002). The same technique has also been applied
to compare the genome of B. anthracis with 19 strains
of related Bacillus species, such as B. thuringensis and
B. cereus (Read et al., 2003). Previously, a comparative genome analysis among B. anthracis and other
non-pathogenic Bacillus species had been restricted
to the virulence plasmid pXO1 for the identification
of potential vaccine candidates (Ariel et al., 2002).
A similar concept has been applied to the identification of antigens conserved in M. tuberculosis and
M. bovis, but absent from the Pasteur vaccine strain.
In this case, bioinformatics tools have been used to
identify genes conserved in the two species, but which
had been lost from the vaccine strain during evolution
(Cockle et al., 2002).
To analyze the genome plasticity in pathogenic
and commensal E. coli isolates, Dobrindt et al.
made use of a whole-genome approach. Using DNA
microarrays, the presence of all translatable ORFs of
non-pathogenic E. coli K-12 was investigated in 26 extraintestinal and intestinal pathogenic E. coli isolates,
three pathogenicity island deletion mutants, commensal and laboratory strains. In addition, they developed
an E. coli pathoarray, which consists of hundreds
probes specific for virulence-associated genes of
ExPEC, IPEC and Shigella, in order to evaluate the
distribution of these genes among the pathogenic and
commensal strains used (Dobrindt et al., 2003).
Examples of comparative proteomics strategies include the study performed between M. tuberculosis
strains of M. bovis BCG strains, with the idea that antigens present only in the virulent strain will be potential vaccine candidates (Jungblut et al., 1999, 2000;

Mattow et al., 2001). The authors identified 96 spot

differences between the strains when they analyzed
whole-cell lysates. Fifty-six spots were found to be exclusive to M. tuberculosis, of which 32 were identified
by mass spectrometry. Furthermore, a very recent work
has been published on H. pylori (Govorun et al., 2003).
In this study, proteome maps of four H. pylori clinical
isolates were obtained using 2D-electrophoresis and
MALDITOFmass-spectrometry. In order to evaluate
their potential as suitable vaccine candidates, the variability of some H. pylori proteins and the level of their
expression in the isolates have been evaluated. Similar approaches for the identification of putative antigens produced by this pathogen had been performed
previously using both DNA microarray (Salama et al.,
2000) and comparative proteome analysis (Jungblut et
al., 2000).
In the last years, the comparative genome and proteome analysis has been widely applied to many other
pathogenic species such as S. pneumoniae (Oggioni
and Pozzi, 2001), Streptococcus pyogenes (Smoot et
al., 2002), L. monocytogenes (Buchrieser et al., 2003;
Glaser et al., 2001), Campylobacter jejuni (Pearson
et al., 2003) and Yersinia pestis (Hinchliffe et al.,
2003).

8. Conclusions
Genomics has introduced a new paradigm in approaches to bacterial pathogenesis. Instead of dissecting bacterial components in vitro, the new approach starts with the complete information on the
genome and on the gene products and then identifies among these the important factors in virulence.
Moreover, the availability of complete genome sequence information on many pathogens has led to a
new paradigm in vaccine development. If a suitable
assay is available, every protein synthesized by the
pathogen can be tested as a vaccine candidate without any prior selection based on incomplete knowledge of the pathogenicity and immunogenicity of the
organism.
Compared to conventional microbiological approaches, the genome analysis of MenB has allowed
the identification of a higher number of novel surfaceexposed proteins, which are highly conserved among
distantly related strains and are also able to induce bac-

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

tericidal antibodies. These novel antigens hopefully

will be the basis for the clinical development of a
vaccine not only against group B Meningococcus, but
also against other serogroups and species of pathogenic
Neisseriae.
Many infectious diseases are still waiting for effective vaccines to be developed. The publication of the
complete genome sequence of many bacteria, parasites
and viruses means that the reverse approach to vaccine
development can be put into practice. In this new perspective, we would expect that the number of candidate
antigens for new vaccines to increase radically in the
near future, therefore promising more ways to combat
bacterial infections.

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