MICROBIAL GENOMICS: TARGETED ANTIMICROBIAL THERAPY AND GENOME VACCINES
Since its pioneering introduction in 1796 by Edward Jenner, vaccination has been revealed as the most effective medical intervention for the prevention of human infections, greatly contributing to increased life expectancy(1). Formal vaccine development started only one century later, when it became clear that infections were caused by microbes, and Louis Pasteur proposed to “isolate, inactivate and inject the microorganism.” This practice established the basis for further key interventions by Jonas Salk and Albert Sabin leading to the eradication of poliovirus infections, and by Maurice Hilleman, who developed vaccines against measles, mumps, and rubella(2). At that time, vaccination approaches were mainly based on the use of crude inactivated or attenuated whole microorganisms.
In the first half of the twentieth century, Glenny, Ramon, Pappenheimer, and others pioneered the isolation and partial purification of bacterial or viral culture components, paving the way for the development of subunit vaccines like those against diphtheria, tetanus, and influenza. Fifty years later, the vaccine field greatly benefited from the introduction of new technologies such as antigen production by recombinant DNA approaches, chemical conjugation of proteins to polysaccharide antigens, and the use of novel adjuvants. New vaccines against important pathogens like Neisseria meningitidis, Streptococcus pneumoniae, Haemophilus influenza, and more effective adjuvanted vaccines were developed. In less than a century, vaccines based on Pasteur’s principle had an enormous impact on global public health by globally eliminating some of the most devastating infectious diseases(3). Table 11.1 lists the vaccines licensed to date. However, it also became apparent that new technologies were required to defeat a large number of diseases still causing high morbidity to mankind, including tuberculosis, malaria, HIV, hepatitis C, Group A and B streptococcal infections, emerging diseases like pandemic influenza, as well as a growing list of infections caused by antibiotic-resistant bacteria. In 1995, the genomics revolution was about to start with the completion of the genome sequence of Haemophilus influenzae(4), opening a new era in vaccine development.
Table 11.1 LICENSED VACCINES FOR HUMAN USE
SOURCE: Horizon Scientific Press/Caister Academic Press UK
DISCOVERY AND DEVELOPMENT OF GENOMIC VACCINES
The recent advances in microbial and human genomics have greatly accelerated the development of novel tools for the diagnosis, monitoring, prevention, and treatment of human infectious diseases (Figure 11.1). During the twentieth century, the invention and development of vaccines have been the major goals of microbiology and preventive medicine, encompassing the whole field of vaccinology. While historically most vaccines were developed using the target microorganism itself, the current focus is utilizing the microbial genomes for developing the targeted vaccine by an approach called “reverse vaccinology.”
Figure 11.1 Impact of the recent advances in microbial and human genomics on the development of novel tools for the diagnosis, monitoring, prevention, and treatment of human infectious diseases.
THE FIRST GENOME-BASED VACCINE: MENINGOCOCCUS B
Once the full set of genes of a bacterial pathogen could be made available, the possibility emerged to identify vaccine targets by computer-facilitated predictions of antigen surface exposure and immunogenicity, without the need of cultivating the pathogen. The genes encoding potentially suitable vaccine targets could be expressed and purified by high-throughput methods in non-pathogenic hosts, and tested in preclinical models for their immunogenicity and their ability to neutralize the original infectious agent.
Figure 11.2 Reverse vaccinology approach for the identification of vaccine candidates starting from the genome sequences of the pathogen.
The first successful application of this novel reverse vaccinology strategy (Figure 11.2) came from the work by Pizza and coworkers on Neisseria meningitidis serogroup B (MenB)(5),(6). MenB, causing 50% of the meningococcal meningitis worldwide, had been refractory to vaccine development due to the identity of its capsular polysaccharide to a human self-antigen and to the extreme variability of its major outer membrane proteins. The MenB genomics vaccine project started with full DNA sequencing of the virulent strain MC58 and bioinformatics analysis of its 2,158 open reading frames (ORFs). Based on prediction algorithms, 570 ORFs were expected to encode surface-exposed or secreted proteins that might be accessible to the immune system. Further steps towards the selection of the best vaccine candidates comprised expression of the predicted antigens as recombinant proteins in Escherichia coli (350 ORFs), assessment of their high exposure on the meningococcal surface (91 candidates), and testing of their ability to elicit antibodies mediating MenB killing by serum bactericidal assays and/or protection against lethal challenge in an animal infection model (28 selected candidates). After screening these 28 antigens on a panel of diverse isolates to determine whether their sequence was well conserved, a multi-component vaccine was finally selected for development. This genome-based MenB vaccine consists of three recombinant proteins representing five MC58 antigens, plus outer membrane vesicles derived from another MenB isolate(7). Notably, the main vaccine antigens were identified as important virulence factors(8). Factor H binding protein (fHbp) binds a key inhibitor of the complement alternative pathway enabling the meningococcus to evade killing by the innate immune system; the Neisserial heparin binding antigen (NHBA) also plays a role in serum resistance; and the Neisserial adhesin A (NadA) mediates bacterial adhesion to host cells.
Further molecular epidemiological investigations revealed a certain degree of sequence variability in the vaccine antigens expressed by MenB isolates obtained from different patients. Antibody cross-reactivity was demonstrated between the NHBA variants and also between the three main NadA variants identified in hyper-virulent strains. Little cross-protection was instead observed between the fHbp variant present in the MenB vaccine and the other two fHbp variant groups. Additionally, the level of expression of all MenB antigens was shown to vary between strains. For these reasons, a new typing system (the meningococcal antigen typing system, or MATS) was developed to predict potential vaccine coverage among infective isolates in different geographical settings(9). MATS is a sandwich enzyme-linked immunosorbent assay (ELISA) that measures the amount of each target antigen expressed by a strain and its immunological cross-reactivity with the protein variant present in the MenB vaccine. The data obtained with MATS correlate with the killing of strains in a serum bactericidal activity assay, and allowed the prediction of 78% coverage of the European MenB isolates. Results from clinical trial studies have shown safety and robust immune responses in both adults and infants(10), forming the basis for the recent licensure of this vaccine by the European Medicines Agency.
ADDRESSING ANTIGEN VARIABILITY BY MICROBIAL COMPARATIVE GENOMICS
Emerging technologies have greatly accelerated genome sequencing during the past decade, leading to a further evolution of reverse vaccinology to incorporate comparative in silico analysis of multiple genomes from different strains of the same species. This approach allows the selection of vaccine antigen candidates, while taking into account the intra-species antigen diversification stratagem adopted by many pathogenic species to escape the immune system. Intra-species diversity is generated by a variety of mechanisms, including mutation, horizontal gene transfer through mobile genetic elements and recombination(11).
Multigenome reverse vaccinology was first applied to Streptococcus agalactiae (Group B streptococcus, or GBS), a Gram-positive microorganism that colonizes the ano-genital tract of 20–30% of healthy women and is a major cause of neonatal sepsis and meningitis. The pathogen can also cause severe invasive infections in the elderly, in pregnant women, and in patients with underlying disease(12). There are ten GBS serotypes distinguished by their capsular polysaccharide, and the amount of maternal antibodies directed against each polysaccharide type is inversely proportional to the risk of neonatal infection with strains of that specific serotype. This observation established the basis for the development of vaccines based on capsular polysaccharides conjugated to carrier proteins, which induce long-lasting immune responses(13).
Parallel efforts to identify protective protein antigens capable of conferring wide coverage were also undertaken, given that protection by GBS polysaccharides is serotype-specific and that non-typeable isolates not expressing any capsule cannot be protected against by polysaccharide-based vaccines. Analysis of the full genome of eight different GBS strains by Tettelin et al. revealed novel genes to be added to the species gene pool after each strain was sequenced. This observation highlighted GBS intra-species diversity and introduced the concept of the “pan-genome,” which comprises “core” genes shared by all strains, and “dispensable” genes present only in one or a few strains(14). Maione and colleagues applied the pan-genome notion to design a universal vaccine against GBS(15). By computational analysis of the eight sequenced genomes, they predicted 589 surface-exposed proteins, 396 of which encoded in core genes and 193 in dispensable genes. Of these 589, 312 were successfully expressed as recombinant proteins in E. coli and evaluated for their ability to mediate protection in a mouse ‘maternal immunization–neonatal pup challenge’ model. A four-antigen combination proved protective against a large panel of strains. Three of these protective antigens were encoded in dispensable genes, and would not have been identified if only a single genome had been screened. Interestingly, these three proteins were seen to assemble into previously undescribed long filamentous pilus-like structures extending outside the bacterial surface that were shown to play an important role during bacterial infection. Subsequent genomic analysis using a wider collection of strains revealed three different pilus islands, PI-1, PI-2a, and PI-2b, at least one of which was present in a wide panel of strains. More interestingly, a vaccine incorporating one component of each pilus variant was shown to provide a high level of mouse protection against virulent isolates representing all GBS serotypes(16).
A similar comparative genomics approach was successfully applied to Streptococcus pneumoniae (pneumococcus), another major human pathogen causing sepsis, meningitis, pneumonia, otitis media, and sinusitis, which accounts for more than 10% of the mortality worldwide in children under five years old(17). Pneumococcus can be classified into more than 90 capsular serotypes, and the recently introduced polysaccharide-conjugate vaccines have proven highly effective in preventing pneumococcal infections against their represented serotypes(18). Pneumococcal protein antigens have additionally been evaluated for their use in universal serotype-independent vaccines to face variable regional distributions of serotypes, the occurrence of serotype replacement after vaccination, as well as the complexity and cost of conjugate vaccines(19). The availability of multiple pneumococcal genome sequences, combined with an increased understanding of pili in GBS and in other Gram-positive pathogens, led to the discovery of pneumococcal pilus proteins eliciting high protection in mouse infection models as potential components of a broad-coverage vaccine combination(20).
A further step in multi-genome reverse vaccinology introduced an additional criterion for the selection of antigens specific to pathogenic strains and absent in commensal strains of the same species, with the aim of reducing the potential impact of a vaccine on the commensal flora. Comparative analysis of the genomes of two E. coli strains causing meningitis, five uro-pathogenic strains, one avian strain, and the non- pathogenic K12, identified 230 surface antigens present in the extra-intestinal pathogenic E. coli but absent (or poorly conserved) in the non-pathogenic isolate. Nine potential vaccine antigens were able to induce protection in a mouse-challenge sepsis model, some of which also present in intestinal pathogenic E. coli, showing promise for a broad-coverage vaccine against different pathogenic E. coli(21).
Genomic reverse vaccinology approaches have now been applied to the discovery of new vaccines against many other pathogens, including Chlamydia pneumoniae, Bacillus anthracis, Porphyromonas gingivalis, Mycobacterium tuberculosis, Helicobacter pylori, hepatitis C virus, the coronavirus responsible for severe acute respiratory syndrome (SARS), and the malaria parasite Plasmodium falciparum(8). Promising results towards defeating malaria have recently been obtained by vaccinating infants and children with the circumsporozoite protein 1 (CSP-1) fused with the hepatitis-B surface antigen(22).
INTEGRATING GENOMICS, PROTEOMICS, AND IMMUNOMICS FOR VACCINE DISCOVERY
A common drawback of genome-based approaches for vaccine discovery is the need to screen a large number of candidates by laborious and time- consuming in vivo and/or in vitro assays, in order to select a limited number of antigens conferring effective protection. Therefore, several pre-screening strategies aimed at reducing the number of antigens for further biological testing have been attempted. Based on the observation that bacterial vaccines inducing protective antibodies are mainly constituted by highly expressed surface-exposed antigens and/or secreted toxins, proteomic-based methods have been used to selectively identify these categories of proteins. In a pioneering approach, Rodriguez-Ortega et al. analyzed the surface of Streptococcus pyogenes (Group A streptococcus, or GAS), a severe human pathogen for which a vaccine is not yet available, by digestion of live bacteria with different proteases, followed by mass spectrometry (MS) analysis of generated peptides, and identified proteins highly expressed on the bacterial surface and thus accessible to antibodies(23). Similarly, antigens on the surface of Gram-negative bacteria were identified by MS analysis of membrane fragments released by the bacteria upon genetic modifications to weaken their outer membrane(24).
An alternative approach aimed at reducing the number of antigens in the pre-selection stage consists of interrogating the entire antigenic repertoire of a particular pathogen by using representative libraries of recombinant peptides that can be displayed on the surface of bacteria or bacterial phages, or spotted onto microarrays. These libraries can then be screened with sera from infected individuals who recovered from infection for the presence of specific antibodies, leading to the identification of a discrete number of antigen targets(25).
In a recent study, Bensi et al. devised a strategy that incorporates quantification of bacterial surface proteins using antibodies raised against recombinant surface-predicted antigens, MS proteomic analysis, and high- throughput screening of human sera, for the rapid selection of a limited number of vaccine candidates prior to biological testing. By applying this combined approach to GAS, highly selective identification of few protective antigens was achieved, which allowed the definition of a multi-protein formulation conferring consistent protection against multiple GAS serotypes in mouse models of infection(26).
The new information derived from the growing list of protective antigens from different microbial species is expected to bring an additional improvement to the prediction of vaccine candidates by bioinformatics tools. Indeed, several curated databases have been established, based on the information obtained from experimentally validated antigens. Ultimately, improved algorithms are expected to be developed that will allow, not only better prediction of surface localization, but also the identification of common signatures among protective antigens, that will guide the identification of novel vaccine candidates.
RATIONAL DESIGN OF EFFECTIVE VACCINES BY STRUCTURE-BASED APPROACHES
Recent advances in X-ray crystallography and nuclear magnetic resonance (NMR) spectroscopy have greatly accelerated structural studies on vaccine antigens and their epitopes, opening the path for the structural design of novel and improved vaccines(27). This type of approach can be applied to address the high variability of many antigen targets of protective antibodies that, as discussed above, is exploited by many pathogens to evade the human immune system.
A structural analysis of MenB fHbp and of the epitopes recognized by protective monoclonal antibodies against the three fHbp variants guided the construction of a chimeric protein with broad protective capacity. The chimera was built by incorporating in variant 1 of fHBP key amino acids from variant 2 and 3 epitopes, while strictly maintaining the three- dimensional structure of the native molecule. To preserve folding, amino acid substitutions were introduced only in residues with side chains well exposed to solvent, leaving the internal core of the protein unaltered.
A similar structural approach was used to obtain a fusion protein covering six variants of the Group B Streptococcus pilus protein 2a. In this case, structural analysis revealed a similar four-domain organization where domain 3 was the main target of protective antibodies. The domain 3 regions from each of the six variants could be fused in a single molecule exhibiting cross- protective properties against strains expressing the different variants.
Structural vaccinology has also been applied to the respiratory syncytial virus (RSV), a virus that infects the lower respiratory tract of most infants and children, and is often associated with hospitalization. RSV is a difficult target for which live attenuated vaccines have been unsuccessful to date, and subunit vaccines, mainly relying on the F glycoprotein antigen, have proven biochemically challenging to develop. F is a trimeric protein that exists in different structural forms; that is, a pre-fusion displayed on infectious virions, a transient intermediate extended structure, and a post-fusion state with detergent-like properties that mediates host cell entry. The F pre-fusion form is the target of most RSV-neutralizing antibodies in human sera, but its instability has hindered its use for a vaccine.
Structural insights guided the engineering of the RSV F post-fusion to a more hydrophilic molecule with increased solubility, where the best- characterized neutralizing epitopes between the pre- and post-fusion forms were well preserved(28). This engineered antigen is now approaching clinical trials for a novel vaccine against RSV. In a second successful study, highly effective prefusion-specific antibodies were identified and used to obtain a co-crystal structure of the antibody in complex with the F glycoprotein locked in its pre-fusion state(29).
The above-described examples clearly indicate that many linear peptides that are usually called epitopes because they are recognized in vitro by antibodies are imperfect mimics of the real surfaces recognized by antibodies in immunized or infected hosts, as they lack a single, defined conformation. Indeed, most often, linear peptides bind functional antibodies with low affinities compared to the native protein or its folded domains, and they might preferentially elicit immune responses against different structural aspects than those recognized by protective antibodies. Therefore, structural vaccinology will be an excellent tool for engineering antigenic surfaces on domains to avoid the use of isolated epitopes.
The development of effective vaccines against the human immunodeficiency virus (HIV) has been hampered by its high antigenic diversity(30). Short peptides representing a wide number of potential T-cell epitopes were selected by computer-based methods and used for the construction of polyvalent vaccine antigens(31). Furthermore, some epitopes targeted by broad neutralizing protective antibodies (bnAbs) towards the HIV envelop protein gp41 have been identified(32). Alternative strategies for eliciting protective B cell responses to HIV focus on trimeric spike protein variants of gp120 that may closely resemble the native spike on infectious virions, although this trimeric structure has not yet been obtained(33). Surprisingly, partial success was attained in a recent HIV vaccine trial in which neither bnAbs, nor potent T cell responses were induced(34).
SYNTHETIC GENOMICS FOR FUTURE VACCINES
Another champion of immune system evasion is the influenza virus, capable of rapidly evolving into thousands of different strains that make necessary the production of a new flu vaccine every year. Most alarmingly, some of the variants can switch hosts, generating new human pandemics.
Finding common universal flu epitopes has been the object of continuous search, and epitopes targeted by cross-reactive human antibodies against hemmaglutinin (HA) were recently identified. These conserved epitopes were found in the stem part of HA, while the variable epitopes used in present vaccines are mainly located in the more exposed head of the protein(30). However, immuno-dominance of variable epitopes remains a key challenge. Deep analysis of the human B cell repertoire and increasingly efficient physical and structural epitope-mapping techniques will help understand the structural basis for epitope immune dominance and improve vaccine design.
While these envisaged results may soon be attained, of capital importance remains the acceleration of the steps leading to the development of each new version of the vaccine necessary to cope with unceasingly arising flu variants. This is particularly true in the case of pandemics, as vaccine prevention tools are required well before the appearance of the peak of disease incidence. Genomic-based approaches have come to our aid in this case also, in the form of “synthetic vaccinology.” In a simulated response to a pandemic with a H7N9 “bird flu,” Dormitzer et al. utilized the viral DNA sequence information to synthesize the genes coding for the necessary HA and neuraminidase (NA) antigen variants(35). The genes were built enzymatically in cell-free reactions that included a critical error correction step. Co- transfection of canine kidney cells with the synthetic genes cloned into optimized expression vectors and plasmid DNA containing improved viral backbone genes produced a combination resulting in high yields of vaccine antigens. The authors could demonstrate the potential of this new procedure to save weeks off the time needed for vaccine manufacture to respond quickly to a sudden pandemic. The study also offered proof of concept for the potential of synthetic vaccinology for the generation of tailor-made microorganisms optimized for the expression of newly designed vaccines.
MONITORING INFECTION EMERGENCE, TRANSMISSION, AND PATHOGEN EVOLUTION BY NEXT-GENERATION SEQUENCING
In this section, we will discuss how genomic-based technologies can be used in clinical microbiology both for the diagnosis and the management of individual infections, as well as for monitoring the emergence and epidemiology of infectious diseases.
GENOMICS IN THE CLINICAL MICROBIOLOGY LABORATORY
The main task of the clinical microbiology laboratory is the rapid management of individual infections by isolating pathogens from clinical samples, identifying species for diagnostic purposes, and testing for antimicrobial susceptibility. Many basic microbiology practices to accomplish these tasks were developed over decades, and are time- consuming and labor-intensive. Most often the pathogen has to be cultured before isolation, and complex selective media are required to treat samples contaminated with colonizing flora. Moreover, diagnostic characterization depends on a wide range of biochemical testing pathways that are often species-specific. This multiple-step process often takes several days, or even weeks in the case of slow-growing microorganisms. The “omics” revolution is expected to deeply transform routine clinical microbiology laboratory practices in the coming years by the progressive substitution of many of these complex multifaceted procedures with genome-based technologies(36),(37).
High-density pan-microbial microarrays containing nucleic acid probes specific for various pathogen sequences now allow the rapid screening of a large number of pathogens: nucleic acids from a clinical specimen can be amplified randomly and then hybridized to the chips for species identification(38).
A proteomics-based approach that has further accelerated species identification consists of comparing mass-spectrometry profiles of pure microbial suspensions with available databases(39). Polymerase chain reaction (PCR) assays can also assist in the rapid identification and typing of bacterial and viral pathogens, and in some cases they have become an integral part of the standard of care. For instance, DNA-sequencing analysis of the HIV genotype for the presence of mutations conferring resistance to anti-retroviral drugs, and PCR-based measurement of viral loads, can be used to choose medication and help predict the responses to therapy(40), and similar approaches have been applied to other viral diseases like hepatitis B and C and influenza. As far as testing of antimicrobial susceptibility in concerned, checking for the presence or absence of antimicrobial resistance genes by PCR can accelerate effective treatment of infected patients.
Application of next-generation full-genome sequencing will soon be sufficiently fast, accurate, and cheap to be routinely used by the clinical laboratory for improved patient care. The major advantage of whole-genome sequencing is that comprehensive DNA information can be obtained in a single rapid step, providing all necessary data for diagnostic and typing needs. Nevertheless, substantial challenges will need to be overcome before microbial full-genome sequencing can be fully embraced in a clinical laboratory setting and gradually replace present-day methodologies. Success will depend on the development and implementation of reliable and user- friendly bioinformatics tools to rapidly extract the most meaningful genetic information from the generated complex data sets.
TRACING INFECTION OUTBREAKS AND TRANSMISSION EVENTS
Microbial genomes are much smaller than eukaryotic genomes but much more diverse, as up to 40% of their DNA may consist of dispensable sequences that are not shared by all members of the same species. The analysis of single-nucleotide polymorphisms (SNPs) by DNA deep- sequencing allows discriminating between closely related strains of the same microbial species, and has enabled epidemiological studies aimed at reconstructing the source of infection outbreaks and the routes of person-to- person transmission. This type of information can help in managing contemporary threats and preventing future outbreaks(41,(42). By SNP analysis, the geographical origin of historical infections like plague, tuberculosis, or leprosy could be traced back in China, India, and East Africa, respectively, and the sites of their initial spread leading to global dissemination were identified. In a more recent example, genome analysis of Mycobacterium leprae isolated from armadillos identified those animals as a possible source of zoonosis for isolated leprosy cases occurring in the United States.
By understanding the genetic basis of infection outbreaks, investigators will be able to validate diagnostic tests to rapidly design appropriate therapies. In addition, future outbreaks can be anticipated, and therefore contained, before becoming widespread. For instance, a panel of 1225 informative SNPs was designed for the rapid typing of entero-hemorragic E. coli O157:H7 isolates during outbreaks.
Different microbial isolates of the same species can vary in their capacity to cause disease according to the presence and expression of genes encoding toxins, adhesins, and drug resistance, often carried by mobile genetic elements like prophages. Intra-species whole-genome sequencing allows discriminating between isolates with different pathogenic potential, and evaluating their genetic relationships to infer the sequence of events driving pathogen evolution(41),(42).
Longitudinal genomic studies performed on different isolates of Streptococcus pyogenes demonstrated that strains causing invasive disease are tightly related to those causing mild oro-pharyngeal infections, and differences in virulence, disease phenotype, and epidemic behavior are probably due to genes encoded on mobile elements rather than on the core chromosome. Furthermore, evidence for adaptation in genes involved in virulence regulation supported a model in which mutation in vivo plays an important role in progression from mild to severe S. pyogenes invasive disease. The authors could indeed demonstrate that single-nucleotide mutations affecting the production of a secreted protease implicated in tissue destruction and dissemination could significantly change the necrotizing fasciitis capacity of particular subclones, thus offering new targets for therapy and vaccine design(42).
MONITORING THE EFFECT OF MEDICAL INTERVENTION ON PATHOGEN EVOLUTION AND DETECTING WITHIN- HOST MICROBIAL VARIATION
A deeper understanding of the microbial population structure may also help researchers monitor the effect of public health interventions, such as antibiotic use and vaccine introduction, on pathogen evolution.
The recent discovery showing that genome-wide mutation rates in latent tuberculosis infection are similar to those occurring in active disease may explain why monotherapy for patients with latent infection is a risk factor for selecting isoniazid-resistant strains(43). Emergence of the community-acquired, methicillin-resistant Staphylococcus aureus (CA-MRSA) USA 300 clone in the United States and throughout the world is a major public health concern. By analyzing the genome sequences of 10 CA-MRSA isolates recovered from diverse regions, Kennedy and collaborators demonstrated a single clonal lineage undergoing expansion and diversification(44). The data suggest that the CA-MRSA clone will continue evolving under host-selective pressure and that higher-virulence clones may arise, further emphasizing the need for a preventative vaccine. Harris et al. used population genomics to trace person-to-person transmission of CA-MRSA strains within a hospital, confirming the potential of this approach to identify unrecognized transmission chains for nosocomial infection, determine the point source, and precisely guide infection-control activities.
The emergence of non-vaccine strains in Streptococcus pneumoniae has been a concern since the introduction of the heptavalent conjugate polysaccharide vaccine in 2000, as it protects against many, but not all, serotypes. Genomic studies have found evidence for capsular switching, in which hybrid strains arising through recombination and expressing non- vaccine capsular types increased in their frequency due to vaccine selective pressure(45).
Many human pathogens are common constituents of the normal flora, and their evolution during colonization may trigger a transition from healthy carriage to invasive disease. Whole-genome sequencing in populations of bacteria colonizing a single individual is shedding light on the microbial evolution dynamics within the host(41). Genetic variation, including SNPs, short insertions and deletions (indels), and mobile elements, has been discovered in single human hosts colonized by species as disparate as Mycobacterium tuberculosis, Salmonella enterica, and Staphylococcus aureus. In a study on a long-term carrier of S. aureus who developed a bloodstream infection, the genomes of invasive bacteria were found to possess an excess of mutations that truncated proteins, including a transcriptional regulator implicated in pathogenicity. Another study, of a 16- year outbreak of chronic Burkholderia dolosa infection, revealed evidence for parallel adaptive evolution of 17 genes across 14 cystic fibrosis patients.
METAGENOMICS AND THE HUMAN MICROBIOME
The different sites of the human body are populated by complex microbial communities, which have become the subject of a new field in microbiology aimed at defining the “microbiome” composition, its interactions with the human host, and its role in human health(46). Given the impossibility of cultivating most of the bacteria composing the human microbiome, broadly applicable techniques for analyzing massive amounts of DNA sequence data have been developed, which have contributed significantly to the growing field of metagenomics. These studies have demonstrated great variations between host individuals and confirmed that substantial alterations in the human microbiome are important for a variety of disease states, including psoriasis, sexually transmitted infections, Crohn disease, gastroesophageal reflux disease, and others. Recent studies have also addressed the role of the gut microbiome in the development of the immune system. The established links between microbial communities and the etiology of human disease will inform future design of better vaccines and therapeutics.
CONTROLLING BACTERIAL INFECTION BY GENOME- BASED ANTIMICROBIALS
Most of the antibiotics in use today originated many decades ago as natural products isolated from bacteria and fungi. The antimicrobial industry has excelled at fine-tuning these natural molecules to improve their spectrums, efficacy, and safety, especially by means of semi-synthetic chemistry approaches. Nonetheless, the growing emergence of antibiotic-resistant bacterial strains and the public health threat of pandemic viral infections have recently raised a renewed interest in the discovery and development of novel non-toxic and fast-acting antimicrobial drugs. The Infectious Disease Society of America estimates that 70% of hospital-acquired infections in the United States are resistant to one or more antibiotics.
GENOMICS AND TARGET-BASED ANTIMICROBIAL DISCOVERY
Genome-based approaches applied at the early stage of the drug discovery process have generated a valuable inventory of genes and cellular processes from which to further test and validate novel antibacterial targets(47). Most of these targets are selected for their essential role during in vitro growth, usually by means of genetic manipulation (e.g., gene knockout) of the relevant bacteria. An alternative approach for target selection focuses on virulence factors required by specific bacteria to cause disease, like toxin delivery or cell adhesion, and has the potential advantages of better preserving the host microbiome and of decreasing the probability of antibiotic resistance(48). Novel antimicrobial targets can also be selected by “metabolomics” approaches—that is, analysis of metabolite production by host cells by NMR or Mass Spectrometry(49). For instance, changes in the metabolic flux of human cytomegalovirus–infected cells were used to identify metabolic pathways upregulated by viral infection, and potential targets for novel antivirals aimed at blocking viral replication.
By comparative genomics, target genes can be selected for narrow- or large-spectrum therapeutic solutions on the basis of their conservation profiles across species. Once validated, these target genes can be cloned and sequenced, and their protein products expressed in an optimized expression system (e.g., Pichia pastoris, Baculovirus, E. coli). Targets are often screened by high-throughput methods against large libraries of combinatorial chemistry-derived compounds(50),(51). Each specific molecular target or pathway of interest is combined systematically with each possible drug compound by automated platforms, and positive results, or “hits,” are subsequently characterized with respect to potency, mechanism of inhibition, spectrum, and selectivity(36),(52). An example of an antimicrobial target identified by a genomics-driven approach is the product of the def gene, which is present in all pathogenic bacteria and does not share a functionally equivalent gene in mammalian cells. The gene encodes a peptide deformylase belonging to the matrix metallo-protease family of enzymes, and a selective inhibitor could be identified by screening a library of metallo-enzyme inhibitors.
An important condition that dictates the efficiency of antimicrobial compounds is their need to cross the microbial membrane barrier and to avoid subsequent extrusion by multidrug-resistance efflux pumps. One strategy that takes into account permeability and efflux issues consists of combining genomics with classic whole-cell screening methods by using genetically modified microorganisms that can respond in a measurable manner when a target of interest is inhibited. The response can be determined as growth inhibition (absorbance) or induction of a linked reporter gene (e.g.,luminescence or fluorescence)(52).
A promising novel group of anti-infectives is represented by the family of lysins naturally produced by bacterial viruses (bacteriophage) to digest the Gram-positive bacterial cell wall for phage progeny release. A large variety of phage enzymes with different specificities was identified by genome analysis and successfully used in animal models to control antibiotic-resistant bacteria on mucosal surfaces and in blood. The advantages over other antibiotics reside in pathogen specificity without disturbing the normal flora, low chance of bacterial resistance to lysins, and their ability to kill colonizing pathogens on mucosal surfaces(53).
The incorporation of automation, computational methods, and nanotechnology has allowed for greatly increased efficiency in the development of both combinatorial libraries and high-throughput screening of potentially useful drugs. Nevertheless, to date, only a few candidates against genetically validated bacterial drug targets derived from these types of screens have attained clinical testing. Structural characterization of inhibitor–target complexes is expected to further assist the design of higher- affinity drugs with improved pharmacological properties. We are currently witnessing an explosion in technological and computational advances in structural genomics, with protein structures of hundreds or thousands of medically relevant targets from infectious disease organisms likely to be available over the next few years. This new information is expected to provide an unprecedented opportunity to accelerate the development of new and improved chemotherapeutic antimicrobial agents.
ANTI-INFECTIVE MONOCLONAL ANTIBODIES
Human genomics now enables the examination of the full epitope repertoire of antibodies in infected and in vaccinated individuals, which can facilitate the development of therapeutic monoclonal antibodies (mAbs). The strategy of displaying human antibody fragments on phage surfaces has produced several mAbs with potential therapeutic applications against agents of infectious disease, including influenza A virus, Clostridium difficile, HIV, viral hepatitis, rabies, Pseudomonas aeruginosa, methicillin-resistant S. aureus, and Bacillus anthracis(54). Of these, the mAb targeting the protective antigen of B. anthracis, raxibacumab, has met all criteria for approval by the U.S. Food and Drug Administration(55).
A very promising approach for the discovery of novel therapeutic antibodies and vaccine targets consists of isolating single B cells from patients having recovered from a particular infection, followed by deep sequencing of single B cell clones and production of human monoclonal antibodies with neutralizing or opsonizing capacities(56).
Interrogation of the entire human B cell response to infection or vaccination has also been applied to the identification of cross-reactive protective epitopes that may represent structural determinants of broadly protective vaccines to overcome high viral mutation rates. This process has been named “analytical vaccinology,” and it was made possible by several recently developed methods for generating human monoclonal antibodies from blood samples(57), which are tested for broad neutralization in high- throughput functional assays(58).
HUMAN GENOMICS AND PERSONALIZED MEDICINE TO COMBAT INFECTIOUS DISEASES
As for other fields of medicine, the recent advances in human genome sequencing and computational biology are expected to revolutionize the treatment and prophylaxis of infectious diseases. Applications of personalized genome-based medicine to the prediction of individual factors that can predispose or affect the response to certain infections as well as individual responses to therapeutic and prophylactic measures are already becoming a reality(36).
The first evidence that certain infectious diseases have a genetic predisposition initially came from a study showing an increased risk of mortality in children born to parents who also died from an infection(59). Since then, defects in genes encoding effectors of the immune response have been associated with an increased susceptibility to certain infections, like those caused by S. aureus and M. tuberculosis. High-density DNA arrays capturing human genome-wide variation can be used to analyze the association of certain SNP with susceptibility to different bacterial and viral diseases(60).
Genome-wide association studies (GWAS) have also identified a series of markers associated with the quality of individual responses to disease treatment(36). For example, a SNP located upstream of the gene encoding the type III interferon was found to be associated with differences in the extent of response to an anti-HCV (human C virus) drug treatment, while IL28B gene polymorphisms were associated with the spontaneous clearance of acute HCV. Large-scale profiling of the full RNA of peripheral blood mononuclear cells (PBMCs) by microarrays or deep RNA sequencing approaches has been applied to the identification of patterns of gene expression characteristic of certain human infections like tuberculosis, dengue, influenza, S. aureus and salmonellosis, among others. Data derived from these approaches will assist the diagnosis and prognosis of disease, the design of antiviral and antibiotic therapies, and the development of genetic tests to predict adverse reactions to antimicrobial drugs.
In the vaccines field, novel tools of systems biology can be applied to the analysis of human immunological response patterns to vaccines in order to uncover molecular signatures of vaccine efficacy and guide the design and evaluation of new vaccines(61). This “systems vaccinology” strategy has been applied to examine the initial molecular signatures in individuals vaccinated against yellow fever, or after administration of the trivalent inactivated influenza virus vaccine. Similar approaches have been used to study immune responses to Brucella melitensis and fungal infections. The obtained data will ideally lead to the design of vaccines capable of inducing optimal immune responses without toxic effects, thus improving vaccine safety profiles.
Integration of increasingly complex high-throughput data into descriptive and predictive equations for immune responses to vaccines is expected to drive faster and more accurate ways of screening vaccine candidates for their effectiveness. Pulendran et al.(62) have predicted the development of a vaccine chip microarray, similar to the MammaPrint prognostic chip that was developed for breast cancer, which will be able to predict the immunogenicity of any vaccine.
Like in other genomic fields, the successful clinical application of these novel technologies will depend on the development of translational research approaches capable of dealing with the enormous amount and different types of generated information and with the uncertainty that is typical of common clinical scenarios.
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