Impact of Genomics on Vaccine Design

Despite advances in the treatment of infectious diseases, pathogenic microorganisms are the single most important threat to health worldwide. Approaches to vaccine development have made remarkable progress in the last 200 years, and vaccination has prevented illness and death for millions of individuals every year. 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.

Most of the vaccines currently available are based on killed or live-attenuated microorganisms, toxins detoxified by chemical treatment or site-directed muta-genesis, purified antigens, or polysaccharides or oligosaccharides conjugated to proteins. Knowledge of the pathogenesis of many microorganisms, the identification of the main virulence factors, and characterization of the immune response after infection have been fundamental to the design of second-generation vaccines mainly based on highly purified antigenic components [1].

The first important innovation in the vaccine field was the introduction of modern molecular biology and microbiology techniques. This approach generated two efficacious recombinant vaccines: the hepatitis B vaccine, which is based on a highly purified capsid protein [2], and the acellular vaccine against Bordetella pertussis, based on three highly purified proteins, including a genetically detoxified toxin [3, 4].

The conventional vaccinology approach requires the pathogen to be grown in laboratory conditions in order to produce individual components in a pure form and sufficient amounts to be tested for its ability to induce an immune response. There are many limitations to this approach: it is time-consuming, it is not applicable to noncultivable pathogens, and in many cases the antigens expressed in vivo during infections are not produced under laboratory conditions, or are variable in sequence.

A second revolution in vaccine design started with the genomic era. The complete genome sequence of a bacterium can be obtained in a brief period of time using the "shotgun sequencing strategy." By means of this technique, in 1995 the first complete genome sequence for a free-living organism (Haemophilus influenzae) was obtained by The Institute for Genomic Research (TIGR; http://www. tigr.org) [5]. In the last few years the number of available genomes has grown considerably (Fig. 24.1). It is now possible to determine the complete genome sequence ofa pathogen in a short period oftime (months) at low cost.

To date, 175 bacterial genomes have been completed and published, and nearly 500 other microorganisms are being sequenced (GOLD Genomes OnLine database at http://www.genomesonline.org/). This panel of bacterial genomes already covers most of the pathogens impacting heavily on human health and therefore of interest for vaccine researchers.

As genome sequences become available, it is now possible to compare related bacteria and pathogens against commensals of the same or related species and even bacteria with different or similar pathogenic profiles, identifying putatively disease-related genes (comparative genomics). 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, isoelectric point (pI), and solubility. Moreover, a putative function can be assigned to each open reading frame (ORF) on the basis of 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. One of the most interesting applications of the genome analysis of pathogenic bacteria is the screening of the inclusive set of proteins potentially encoded by a microorganism in search of potential vaccine candidates, regardless of their abundance or expression conditions. This new approach has been termed "reverse vac-cinology" [6], indicating that, in contrast to conventional vaccinology, the starting point for vaccine design is the in silico analysis of the genome sequences and not the live bacterium (Fig. 24.2).

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 genome-proteome technologies.

In this chapter we will describe how genomic information has been successfully used to identify novel potential vaccine candidates against various human pathogens, and illustrate the application of functional genomics to vaccine research.

Fig. 24.1 A representative list of available bacterial genomes, showing the rapid increase that has occurred in recent years. The data were obtained from different sources: the TIGR web site (www.tigr.org), the Sanger web site (www.sanger.ac.uk), the NCBI web site (www.ncbi.nlm.nih.gov/ PMGifs/Genomes/micr.html), and the GOLD Genomes OnLine database (www.genomesonline.org).

Fig. 24.2 Application of reverse vaccinology to identify vaccine candidates, showing the different steps of this approach, starting from in silico analysis of the genome (a), to selection of potential vaccine candidates (b),

cloning and purification ofthe selected antigens (c), and analysis ofthe immune sera in vivo or in vitro for the evaluation of the best candidates to be considered for vaccine development (d).

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