445

Reverse vaccinology
Rino Rappuoli
Biochemical, serological and microbiological methods have Figure 1
been used to dissect pathogens and identify the components
useful for vaccine development. Although successful in many
cases, this approach is time-consuming and fails when the Conventional vaccine development
pathogens cannot be cultivated in vitro, or when the most
abundant antigens are variable in sequence. Now genomic Cultivate microorganism
approaches allow prediction of all antigens, independent of
their abundance and immunogenicity during infection, without
the need to grow the pathogen in vitro. This allows vaccine
development using non-conventional antigens and exploiting
non-conventional arms of the immune system. Many vaccines
impossible to develop so far will become a reality. Since the
Test
process of vaccine discovery starts in silico using the genetic Antigen convalescent
selection sera Test
information rather than the pathogen itself, this novel process immunogenicity
can be named reverse vaccinology.
Identify
clone components Purify
genes components
Addresses
5-15 years
IRIS, Chiron S.p.A., Via Fiorentina 1, 53100 Siena, Italy;
e-mail: [email protected]
Immunogenicity
testing in Vaccine Vaccine
Current Opinion in Microbiology 2000, 3:445–450 development
animal models
1369-5274/00/$ — see front matter
© 2000 Elsevier Science Ltd. All rights reserved. 1-2 years

Abbreviations Express
HCV hepatitis C virus recombinant DNA
Men B group B meningococcus proteins vaccines
preparation

Introduction: conventional vaccinology

The conventional approach to vaccine development uses In silico vaccine candidates
two methods: first, attenuation of pathogens by serial Computer
passages in vitro to obtain live-attenuated strains to be prediction
used as vaccines, and second, identification of protective
antigens to be used in non-living, subunit vaccines [1].
In this review, we focus on subunit vaccines. The con- Start from the
whole genomic
ventional way to develop these vaccines is summarized sequence
in Figure 1. In order to identify the components of the
pathogen suitable for vaccine development, the
pathogen is grown in laboratory conditions and the com-
Reverse vaccinology
ponents building the pathogen are first identified one at
a time, by biochemical, serological or genetic methods. Current Opinion in Microbiology
The identification of protective antigens that could be
potential vaccine candidates involves separating each Schematic representation of the essential steps of vaccine development
component of the pathogen one by one. This approach is by the conventional approach and by reverse vaccinology.
time-consuming and allows the identification only of
those antigens that can be purified in quantities suitable
for vaccine testing. Since the most abundant proteins are development is not possible when the pathogen cannot
most often not suitable vaccine candidates, and the be grown in laboratory conditions. An exception to this
genetic tools required to identify the less abundant com- has been the hepatitis B vaccine where the pathogen,
ponents may be inadequate or not available at all, this although unable to grow in vitro, could be recovered in
approach can take years or decades. For the bacterial and large quantities from the plasma of infected people [2].
parasitic pathogens studied to date, the maximum num-
ber of potential vaccine antigens identified during a Once a suitable antigen is identified, it needs to be pro-
century of vaccine development is usually less than ten. duced in large scale, often by growing the pathogen
This conventional method also means that vaccine itself. Cloning of the gene coding for the antigen is often
446 Genomics

necessary in order to better characterize and produce the many of our tissues, is poorly immunogenic and a potential
identified antigen(s). Finally, the new molecule can cause of autoimmunity. On the other hand, the protein-
enter vaccine development. Although successful in many based approach had identified as protective antigens the
cases, this approach took a long time to provide vaccines most abundant proteins of the outer membrane [4].
against those pathogens for which the solution was easy However, these abundant surface-exposed proteins usually
and failed to provide a solution for those bacteria and contain many amphipathic domains, which span the outer
parasites that did not have obvious immunodominant membrane several times and assume a β-barrel conforma-
protective antigens [3•]. tion (Figure 2a). The protective epitopes in these proteins
are located in the loops that are exposed on the external
Reverse vaccinology surface and are usually formed by the precise conformation
The reverse approach to vaccine development takes advan- of a few amino acids. Therefore, in order to induce protec-
tage of the genome sequence of the pathogen. The genome tive immunity these antigens need to be folded within the
sequence provides at once a catalog of virtually all protein outer membrane (recombinant proteins do not induce pro-
antigens that the pathogen can express at any time. As tection) and any change in one of the few amino acids of
shown in Figure 1, this approach starts from the genomic the loop will result in a different epitope. Vaccines based
sequence and, by computer analysis, predicts those anti- on outer membrane vesicles (OMV) and containing the
gens that are most likely to be vaccine candidates. The major outer membrane proteins have been developed
approach can, therefore, be very naïve, and poses the ques- and shown to be efficacious in clinical trials; however,
tion of whether any of the potential antigen candidates can owing to the high sequence variability of the external
provide protective immunity without knowing whether the loops in different MenB strains, protection is induced
antigen is abundant, immunogenic during infection or only against the immunizing strain. As a consequence,
expressed in vitro. This approach allows not only the iden- the conventional approach to vaccine development has
tification of all the antigens seen by the conventional failed to deliver a universal vaccine.
methods, but also the discovery of novel antigens that work
on a totally different paradigm. Therefore, this method Using reverse vaccinology, fragments of DNA were
allows the discovery of novel mechanisms of immune inter- screened by computer analysis while the MenB
vention. The feasibility of the approach relies heavily on nucleotide genome sequence was being determined
the availability of a high-throughput system to screen pro- [5•,6••]. Six hundred novel genes were predicted to code
tective immunity. When this is available, in theory all genes for surface-exposed or exported proteins. These were
of a pathogen can be tested, without any bias of any type. cloned and expressed in Escherichia coli as fusions to the
Unfortunately, owing to our limited knowledge of vaccine glutatione transferase or to a histidine tag. Of these fusion
immunology, good correlates of protection are rare and, proteins, 350 were successfully expressed, purified and
therefore, screening for protective immunity is the rate-lim- used to immunize mice. The sera obtained were used to
iting step of reverse vaccinology. The other limit of this confirm the surface exposure of the proteins by ELISA
approach is the inability to identify non-protein antigens and FACS analysis, and to test for the ability to induce
such as polysaccharides, which are important compo- complement-mediated in vitro killing of bacteria, a test
nents of many successful vaccines, and the identification that correlates with vaccine efficacy in humans. Within
of CD1-restricted antigens such as glycolipids, which 18 months, while the nucleotide sequence was still being
represent new promising vaccine candidates. finalized, 85 novel surface-exposed proteins were discov-
ered and 25 of these were shown to induce bactericidal
Applications of reverse vaccinology antibodies [6••]. These numbers are impressive if one
The publication of the complete genome sequence of considers that during the past four decades no more than
many bacteria, parasites and viruses means that the reverse a dozen of such proteins had been identified. The sur-
approach to vaccine development can be put into practice. prising finding was not only the high number of the new
Below we discuss the different approaches that are being proteins found but also the quality of the new proteins. In
used or potentially could be used to develop novel and addition to the conventional outer membrane proteins
effective vaccines against a variety of pathogens. with variable surface-exposed loops (as in Figure 2a),
many of the new proteins were lipoproteins or other types
Group B meningococcus of surface-associated proteins without membrane-span-
Group B meningococcus (MenB) represents the first ning domains (Figure 2b). These were often conserved in
example of the successful application of reverse vaccinology. sequence, and carried multiple protective epitopes con-
The conventional approach to vaccine development served in most strains. These novel proteins provide an
against this pathogen had been struggling for four decades optimal basis for the development of a novel and effective
without progress. On the one hand, the capsular polysac- vaccine against MenB [6••].
charide used to develop conventional and conjugate
vaccines against all other pathogenic meningococci could Malaria
not be used because the MenB capsule, which is chemi- Malaria, together with AIDS and tuberculosis, belongs to
cally identical to an α2–8 linked polysialic acid present in the triad of the most dangerous diseases that threaten
 Reverse vaccinology Rappuoli 447

human health. The 500 million new infections each year Figure 2
and 2.5 million annual deaths indicate that all measures
used so far to control the disease have failed [7].
(b)
Vaccination would be an effective way to control the
(c)
spread of malaria, but vaccines are not available, despite
many years of research [3•]. Approximately 20 antigens
have been identified from the malaria parasite but none of
them is good enough for a vaccine. The problem is further (a)
complicated by the different antigenic profiles expressed
by sporozoites, merozoites and gametocytes, that the para-
site assumes during its life cycle. The solution can only
come from a genomic approach. The sequence of two of
the 14 chromosomes of Plasmodium falciparum have been
published [8•,9•] and provided the full set of genes con- Outer membrane
tained in the two chromosomes. The complete sequence
of the whole genome will soon provide information on the
predicted 6000 genes. Analysis of the whole genome
expression will show which genes are expressed by the
sporozoite, liver and sexual life-stages of the parasite. Cytoplasm
Expression of genes predicted to be immunogenic as
recombinant proteins delivered with adjuvants or as DNA Current Opinion in Microbiology
vaccines will eventually provide the effective vaccine
against malaria [10••,11].
Examples of protective antigens of Neisseria meningitidis. (a) A
schematic structure of a typical outer membrane protein that is mostly
The task is a formidable challenge, however, it is doable. It embedded within the membrane. These proteins contain one or two
is just a matter of resources and co-ordination. The most dif- protective epitopes located at the tip of most external loops – change of
ficult task is the development of an in vivo or in vitro model one amino acid is enough to escape immunity. Outer membrane proteins
that allows high-throughput screening of vaccine candidates. represent the major part of the antigens of N. meningitidis identified by
conventional vaccinology. Many novel outer membrane proteins have
been identified by reverse vaccinology. (b,c) Schematic structures of
Tuberculosis membrane-anchored lipoproteins or secreted antigens from
Mycobacterium tuberculosis infects approximately two bil- N. meningitidis that have been identified by reverse vaccinology.
lion people worldwide and causes 1.5 million deaths
annually [12]. The inability of AIDS patients to keep the
infection under control and the appearance of multi-resis- Syphilis
tant strains make the disease an unrestrained danger. The During the past four centuries, syphilis has been a night-
available live-attenuated BCG vaccine is not a solution, mare comparable to today’s AIDS [19]. If untreated, this
because of the variable efficacy reported in the trials. sexually-transmitted disease leads to neurological disor-
Furthermore, subunit vaccines have not been developed ders, cardiovascular problems and death, but after the
because all the antigens identified by conventional vacci- discovery of penicillin the disease became easy to control.
nology provide protection that in animal models is lower However, today syphilis represents a new threat both in
than that provided by BCG [12]. Also vaccine develop- developed and developing countries because it causes gen-
ment and testing is complicated by the long time required ital ulcers, which facilitate the spread of HIV. There are
for bacterial growth. approximately 9000 syphilis cases in the US and it is com-
mon in Africa and other developing countries, which are
The sequence of the whole genome of M. tuberculosis [13•] estimated to have 12.5 million cases [20].
has provided a list of all possible genes, which now can all
be expressed as recombinant proteins or as DNA vaccines Syphilis is caused by a bacterium, Treponema pallidum,
and tested for protective immunity [14]. The absence of a that cannot be cultivated in the laboratory and, there-
high-throughput screening for protective antigens makes fore, has been refractory to conventional approaches to
the effort difficult but doable by means of a systematic vaccine development. Attempts to identify vaccine anti-
approach. However, a number of genome- and proteome- gens using the bacterium grown in rabbits had identified
based approaches are providing novel vaccine candidates, approximately 20 different antigens. Once again, the
while at the same time the increased knowledge of this dif- sequence of the complete genome made available at
ficult bacterium makes it easier to approach [15,16,17••]. once all the genes of the bacterium, which can all be
The combination of the genome and the use of the fast- expressed as recombinant proteins or as DNA vaccines
growing Mycobacterium marinum is the winning [21•,22,23••]. Therefore, for the first time it is now pos-
combination to accelerate the discovery of an effective sible to approach development of a syphilis vaccine in a
tuberculosis vaccine [18]. systematic way. The absence of a high-throughput
448 Genomics

Table 1

Comparison of conventional and genomic approaches to vaccine development..

Conventional vaccinology Reverse vaccinology

Essential features
Most abundant antigens during disease All antigens immunogenic during disease
Antigens immunogenic during disease Antigens even if not immunogenic during disease
Cultivable microorganism Antigens even in non-cultivable microorganisms
Animal models essential Animal models essential
Correlates of protection useful Correlates of protection very important
Correct folding in recombinant expression important
High-throughput expression/analysis important

Advantages
Polysaccharides may be used as antigens Fast access to virtually every single antigen
Lipopolysaccharide-based vaccines are possible Non-cultivable microorganisms can be approached
Glycolipids and other CD1-restricted antigens can be used Non abundant antigens can be identified
Antigens that are not immunogenic during infection can be identified
Antigens that are transiently expressed during infection can be identified
Antigens not expressed in vitro can be identified
Non-structural proteins can be used

Disadvantages
Long time required for antigen identification Non proteic antigens cannot be used (polysaccharide, lipopolysaccharides,
Antigenic variability of many of the identified antigens glycolipids and other CD1-restricted antigens)
Antigens not expressed in vitro cannot be identified
Only structural proteins are considered

animal model again makes the problem difficult but not advantage of the knowledge of the genome to design
impossible to solve. totally non-conventional vaccine targets and whether pro-
teins never used in conventional vaccines (i.e.
Hepatitis C virus non-structural proteins) can become effective vaccines.
Hepatitis C virus (HCV) [24] is perhaps the best example These proteins should be able to confer protection mostly
of a vaccine being developed entirely by reverse vaccinol- through cell-mediated immunity and not rely on antibody
ogy. In this case the virus that causes the disease has never neutralization of viral infection. The encouraging results
been cultivated in vitro (it grows only in humans and chim- obtained with some early proteins such as Tat and Rev
panzees [25]) and has never been visualized by electron [30••–32••] in the case of HIV suggest that this may be a
microscopy, making it impossible to use any conventional novel way to protect against viruses.
approach to vaccine development. The cloning and
sequencing of the HCV genome allowed the identification Other pathogens
of the etiological agent [26], the recombinant expression of The pathogens described above are perhaps some of the
its proteins, and the immediate development of diagnostic most representative among those that can be approached
tools, which prevents hundreds of new infections each day by reverse vaccinology. However, the list of the pathogens
ever since. The availability of the genome sequence also where the conventional approaches to vaccine develop-
allowed the prediction of the envelope proteins that nor- ment have failed or provided only partial solutions is
mally are used to develop vaccines against enveloped extensive. Among these we can list bacteria such as
viruses [27]. These proteins (E1 and E2) have been Chlamydia [33•,34,35], pneumococcus [36–39],
expressed in many hosts, but so far only mammalian cells Streptococcus, Staphylococcus, pseudomonas, Borrelia
have been able to express them in a form that induces pro- [40,41••], Escherichia coli, gonococcus, typhoid, Brucella,
duction of antibodies able to interfere with the binding of Ricksettia [42•] and Bartonella (the genome sequences of
E2 to the host receptor [28]. These recombinant proteins most of these pathogens are about to be completed and
have been able to protect chimpanzees from infection with available on the website http://www.tigr.org), and parasites
the homologous HCV virus [29]. such as Leishmania and many others.

While vaccine development using the E1 and E2 conven- Conclusions

tional vaccine targets is making progress, perhaps the Conventional approaches to vaccine development are time
most interesting questions are whether we can take consuming, identify only abundant antigens that may or
 Reverse vaccinology Rappuoli 449

may not provide immunity, and fail when the pathogen 11. Wang R, Doolan DL, Le TP, Hedstrom RC, Coonan KM, Charoenvit Y,
Jones TR, Hobart P, Margalith M, Ng J et al.: Induction of antigen-
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vaccinology (i.e. genomic-based approaches to vaccine vaccine. Science 1998, 282:476-480.
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• Gordon SV, Eiglmeier K, Gas S, Barry CE III et al.: Deciphering the
many pathogens, such as group B meningococcus, has biology of Mycobacterium tuberculosis from the complete
allowed the successful application of reverse vaccinology genome sequence. Nature 1998, 393:537-544.
where conventional approaches have failed. With the The complete sequence of Mycobacterium tuberculosis.

genome sequences of many other bacteria, parasites and 14. Doherty TM, Andersen P: Tuberculosis vaccines: development work
and future. Curr Opin Pulm Med 2000, 6:203-208.
viruses to be completed in the near future, reverse vacci-
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Gordon SV: Genomic analysis reveals variation between
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