- Email: [email protected]
Vaccines to Overcome Antibiotic Resistance: The Challenge of Burkholderia cenocepacia
TIMI 1769 No. of Pages 12
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Review
Vaccines to Overcome Antibiotic Resistance: The Challenge of Burkholderia cenocepacia Viola C. Scoffone,1,3 Giulia Barbieri,1,3 Silvia Buroni,1 Maria Scarselli,2 Mariagrazia Pizza,2 Rino Rappuoli,2 and Giovanna Riccardi1,* Cystic fibrosis (CF) patients are at particular risk of infection by microorganisms that are resistant to several antibiotics. About 3% of CF patients are colonized by Burkholderia cenocepacia, and this represents a major threat because of its intrinsic high level of drug resistance and the lack of a safe and effective treatment protocol. The development of anti-Burkholderia vaccines is a valuable and complementary approach, but only a few studies have been reported to date. In this review we discuss recent advances in the vaccine field and how new technologies, including structural reverse vaccinology, could drive the design of an effective vaccine against B. cenocepacia for use in preventive and therapeutic applications.
Highlights The management of infections caused by multidrug-resistant bacteria is particularly difficult in CF patients, where the complex lung environment in which many pathogens interact complicates testing antibiotic efficacy, leading to increased morbidity. Burkholderia cenocepacia represents a major threat to CF patients because of its intrinsic high level of drug resistance and the lack of a safe and effective treatment protocol, as well as lack of interest by the pharmaceutical industry and scientific journals given the small number of people suffering from this infection.
Antibiotic Resistance and Its Impact on CF Bacterial multidrug resistance is a major challenge of our time: as recently described by the World Health Organization, it represents a global priority, and for some dangerous pathogens a standard eradication protocol is still lacking (https://www.who.int/antimicrobial-resistance/ en/). In recent years many scientists have focused their research on CF patients who are at particular risk of infection by microorganisms resistant to several antibiotics [1]. CF is caused by deficiency in the CTFR (cystic fibrosis transmembrane conductance regulator) ion channel, which leads to the accumulation of dehydrated mucus at the level of different organs, particularly in the lungs, leading to the local proliferation of multiple opportunistic pathogens [2]. The definition and detection of antibiotic resistance is particularly challenging in CF because of the difficulty of recreating the complex CF lung environment where many pathogens interact, and of testing the efficacy of antibiotics in these conditions [1]. This complicates the management of infections and significantly contributes to reduced survival in individuals with CF. One opportunistic pathogen that is particularly difficult to treat is Burkholderia cenocepacia: this represents a major threat because of its intrinsic high level of resistance to drugs currently used in therapy and because of the lack of a safe and effective treatment protocol [3]. Because epidemiological data show that only a small percentage of CF patients (~3%) in the USA suffer from this infection, there has been little investment in the identification of new drugs [4]. In summary, B. cenocepacia is a deadly infectious agent that principally affects people with a rare genetic disease, and there has been limited interest in this opportunistic pathogen from researchers, the pharmaceutical industry, and scientific journals. Diffusion of information regarding this infection is crucial for helping the CF community.
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Vaccine development represents a valuable and complementary approach in the battle against bacterial infections affecting CF patients. However, there have been only a few studies on the development of vaccines against B. cenocepacia [5–10]. In this review we discuss new technological advances in the vaccine field (including reverse vaccinology) that could drive the design of vaccines against B. cenocepacia for use as preventive and therapeutic agents. We also focus on monoclonal antibodies, a new strategy that could be applied to a poorly studied bac-
*Correspondence: [email protected] (G. Riccardi).
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A complementary approach might be the development of vaccines, supported by new advanced technologies in the field (including structural reverse vaccinology), for use as preventive and therapeutic agents.
Department of Biology and Biotechnology 'Lazzaro Spallanzani', University of Pavia, Pavia, Italy 2 GlaxoSmithKline, Siena, Italy 3 These authors contributed equally
https://doi.org/10.1016/j.tim.2019.12.005 © 2019 Elsevier Ltd. All rights reserved.
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terium such as B. cenocepacia. By dissecting the different aspects of B. cenocepacia pathogenesis, antimicrobial resistance, and potential preventive and therapeutic measures against these infections, we aim to raise attention to a deadly pathogen that is a major threat to many CF patients.
Lung Infections in CF Patients Microbial Communities in the CF Lung Environment CF patients suffer from chronic lung infections caused by complex polymicrobial communities that are responsible for inflammation and a progressive decline in lung function (Figure 1, Key Figure). Culture-independent, next-generation sequencing-based studies revealed that known CF pathogens (Pseudomonas aeruginosa, methicillin-resistant Staphylococcus aureus, and multiple species of Haemophilus and Burkholderia cepacia complex) coexist with additional members of the CF lung community – such as Stenotrophomonas maltophilia, Achromobacter spp., nontuberculous mycobacteria (Mycobacterium abscessus complex, Mycobacterium avium complex), the Streptococcus milleri group, and different anaerobes
Key Figure
Overview of the Key Points Described in the Review
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Figure 1. Cystic fibrosis (CF) patients are usually colonized by many bacteria. Of CF patients, 3% are colonized by Burkholderia cenocepacia (in red), for which only a few antibiotics are still effective. The lack of therapies is made worse by the intrinsic resistance of Burkholderia spp. to many antimicrobial compounds via different mechanisms. Putative antigens (mainly with extracellular localization) for vaccine development have already been identified, and the use of new technologies will contribute to the discovery of candidate vaccines with desired characteristics (created with BioRender.com). Abbreviations: EPS, exopolysaccharide; IR-ELISA, immunoreactive ELISA; LPS, lipopolysaccharide; MRSA, methicillin-resistant Staphylococcus aureus; RND, resistance-nodulation-division transporters.
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Box 1. Sources of Infections The airway microbiota is established early in life. The upper airways (nasal and oropharyngeal cavities) are initially colonized by a rich pool of bacteria that are then selected based on their ability to adapt to the new environmental niche. According to the current model, the rich upper airways act as reservoirs of bacteria that can then colonize the lower airway compartments [77]. In this context, the oral cavity represents the main source of bacteria of the lung microbiota. The throat microbiota in healthy and CF individuals is dominated by few genera, including Streptococcus, Veillonella, and Prevotella [16,78]. Interestingly, oral streptococci play a key role in influencing CF community dynamics. Different streptococcal species (Streptococcus milleri group, Streptococcus oralis, Streptococcus mitis, Streptococcus gordonii, and Streptococcus sanguinis) were reported to modulate the pathogenicity of P. aeruginosa [79,80] and to influence the composition of the microbial community by inhibiting P. aeruginosa colonization when streptococci are pioneer colonizers [80]. This observation acquires important relevance when we consider that Streptococcus is one of the most abundant genera in the airway microbiome of CF infants [13]. Although throat and lung show closely related microbiota, the nasal cavity is colonized by a different microbial community [16] that, in healthy adults, is dominated by the genera Propionibacterium, Moraxella, Corynebacterium, or Staphylococcus. CF patients were reported to display a distinct nasopharyngeal bacterial microbiota characterized by an increased abundance of Staphylococcus aureus [81,82]. This CF nasal microbiota pattern is acquired early in life and probably reflects the altered environment of the nasal cavity of CF patients which favors colonization by Staphylococcus while preventing the adaptation and growth of normal commensals. The nasal cavity can therefore represent the origin of S. aureus colonization of the lower airways that is very common in CF patients, especially during infancy. Lung microbial communities are also strongly influenced by the gut microbiota. A high degree of concordance between the gut and respiratory microbial communities was reported in CF infants, and a large number of genera were present in the gut before colonization of the respiratory tract [83]. Finally, some bacterial species that are part of the CF lung microbiota can be acquired from patient-to-patient transmission or from the environment. This is the case of P. aeruginosa and B. cepacia complex bacteria that can be acquired from environmental reservoirs and then spread among patients [84].
such as Prevotella, Rothia, and Veillonella – that could contribute to the disease [11]. The different sources of infections are reported in Box 1. The CF airway microbiota shows a significant interindividual heterogeneity in composition and diversity [12]. During the first 2 years of life, the airway microbial community develops gradually and is enriched in the genera Streptococcus and Haemophilus [13]. Bacterial diversity is greatest in early life until adolescence and then declines with increasing age and disease progression [12,14,15]. This reduction in diversity is generally associated with an increase in the prevalence and dominance of P. aeruginosa [12,16], an extremely virulent, Gram-negative bacterium whose higher abundance was historically considered to be the cause of the onset of pulmonary exacerbations [17]. However, longitudinal studies reported no significant relationship between P. aeruginosa levels and the onset of exacerbations, and showed an unexpected stability of the CF lung microbiota composition [13,18]. In addition to P. aeruginosa, Burkholderia cepacia complex (Bcc) spp. are abundant in the airway communities of CF adults [16,19]. Impact of Antibiotic Treatments The observation that frequent exposure to multiple antibiotics has only a transient impact on the airway microbiota of CF patients highlights the ability of these microbial communities to tolerate antibiotic treatment or to acquire antibiotic resistance. Tolerance to antibiotic can be due to the ability of pathogens to grow in biofilms (P. aeruginosa), to the development of persister cells, and to the establishment of complex interspecies interactions that limit the efficacy of antibiotic treatments [20]. The complexity of these interactions and the interindividual heterogeneity of the microbial communities colonizing the airways of CF patients represent a challenge for the efficacy of therapies, highlighting the need to develop new preventive and therapeutic strategies.
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The Case of B. cenocepacia Why Is B. cenocepacia Difficult to Treat and Eradicate? B. cenocepacia is one of the most clinically relevant members of the Bcc, a group of 24 closely related bacterial species [21], described for the first time by Walter Burkholder in 1949. B. cenocepacia is a ubiquitous nonglucose fermenter aerobic Gram-negative bacterium that is commonly isolated from water, soil, and from sputum of CF patients. The incidence of B. cenocepacia infections in CF patients is only 3%, but this infection leads to a 2.5-fold reduction in their life expectancy relative to noncolonized patients [22]. Indeed, infection can lead to the 'cepacia syndrome', a fatal necrotizing pneumonia characterized by fever and septicemia. CF patients infected by B. cenocepacia have a worse decline compared with patients infected by other CF pathogens such as P. aeruginosa. Moreover, Bcc contamination of medical devices, disinfectants, and pharmaceutical formulations leads to outbreaks among hospitalized non-CF patients such as those undergoing chemotherapy and immunosuppressed individuals. The numerous virulence factors of B. cenocepacia play an important role in its pathogenicity (Table 1). However, this bacterium is extremely difficult to treat and to eradicate because of its high level of antibiotic resistance caused by reduced of cell-wall permeability, increased activity of efflux pumps, mutations in antibiotic target genes, and enzymatic modification or inactivation of the drug (Figure 1). It is noteworthy that the ability to produce biofilm also greatly contributes to antibiotic resistance [3]. Although lung transplantation is an established strategy for end-stage CF patients to improve survival and quality of life, B. cenocepacia infections are reported as a contraindication leading to significantly worse outcome, and 1 year and 5 year survival rates of infected patients are 68% and 42.6%, respectively, versus 93.6% and 73.7% for B. cenocepacia-negative patients [28]. Therapeutic Options for Burkholderia Infections in CF Patients All Burkholderia features described above make it impossible to establish a standard treatment strategy to eradicate chronic infections [29]. The choice of effective antimicrobials is severely limited by the ability of Burkholderia spp. to adapt to adverse conditions including the presence of antibiotics. Indeed, there have been only a few reports of successful eradication treatment in recent years: in one study a child with CF underwent a combination of intravenous (IV) and nebulized antibiotics plus sinus surgery [30]. Garcia and collaborators instead used an intensive
Table 1. B. cenocepacia Virulence Factors
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Virulence factor
Features
Refs
Exopolysaccharide (cepacian)
Phagocytosis and reactive oxygen species (ROS) production interference
[23]
Biofilm formation
Enhanced in the presence of neutrophil-like dHL60 cells; bacterial protection from recognition by the immune system
[24]
Lipopolysaccharide
Infection establishment; neutrophil respiratory burst response and stimulation of the production of proinflammatory cytokines
[23,25]
Secretion systems
T2SS: zinc metalloprotease secretion T4SS-1 and T3SS: role in intracellular survival T5SS: useful for bacterial adhesion T6SS: affects the actin cytoskeleton of macrophages; activates the inflammasome and enhances the activity of caspase-1
[23]
Siderophores
Ornibactin, pyochelin, cepabactin, and cepaciachelin: iron chelation and uptake during infection
[26]
Flagellin
Infection establishment
[27]
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combination of IV, inhaled, and oral antibiotic therapies based on in vitro sensitivities [31]. A 2 week treatment with IV tobramycin, ceftazidime, and temocillin was used, followed by 3 months of inhaled tobramycin, to eradicate Burkholderia infections from two children in the UK [32]. However, the duration of therapy, the use of single or multiple therapy, and the lack of correlation between in vitro and in vivo susceptibility data are still under debate [33]. In addition to the drugs mentioned above (Figure 1), new compounds are in clinical trials, such as the inhaled alginate oligosaccharide (OligoG) whose potential for use in biomedical applications to prevent infections or biofilms has been recently reported [34]. Moreover, the challenge in finding a cure for Burkholderia infections led to the proposal of alternative approaches including (i) molecules already used for other diseases, such as immunosuppressors and corticosteroids, which was successful in two patients [35]; interferon-γ, which until now has been tested only ex vivo [36]; and thiosulfoacid S-esters [37], cysteamine [38], and imidazoles [39] whose activity has been assessed in vitro. (ii) Natural products with antimicrobial activity such as plant nanoparticles [40] that were tested in vitro; fish oils [41], which showed activity in a Galleria mellonella infection model; and glycopolymers [42], that were successfully used against Bcc CF clinical isolates. (iii) Other methods based on the use of phage therapy [43] or antimicrobial peptides that can block B. cenocepacia biofilm formation [44], and quorumsensing inhibitors to attenuate virulence [45]. In this scenario, the development of new therapeutic strategies against this bacterium is extremely important in order to improve the life expectancy of CF patients. Vaccines are the most effective weapons to fight the global rise of antimicrobial resistance because they do not induce selective pressure on the environment and they do not contribute to antimicrobial resistance.
Vaccines against B. cenocepacia Immune Response The characterization of the immune response to B. cenocepacia is extremely complex: CF patients have an abnormal immune regulation because bacteria are embedded in the mucin layer or in the intracellular environment, and this can interfere with the host immune response. The airway epithelium is overlaid by high molecular weight glycoproteins (mucins) that provide a physical barrier. In normal conditions, bacteria bind to the mucins and are cleared by the mucociliary activity. In CF patients, impaired mucociliary clearance leads to the persistence of bacteria in the airway lumen and, consequently, to their access to the epithelial cells. Different surface structures are involved in the interaction between B. cenocepacia and the host lung epithelium: flagella, pili, and adhesins [46]. Furthermore, bacterial lipopolysaccharide (LPS), flagella, pili, and lipopeptides are recognized by the innate immune Toll-like receptors (TLRs) that induce the production of proinflammatory cytokines [47]. B. cenocepacia can survive intracellularly in both airway epithelial cells and macrophages in order to evade host defenses and to establish chronic infections. Flagella and lipases promote invasion of lung epithelial cells, while intracellular survival and replication seem to require other factors such as the type IV secretion system [48]. During cell invasion, bacteria persist in the autophagosomes and replicate in the endoplasmic reticulum, showing that B. cenocepacia is able to subvert the normal endosomal pathway, thus preventing the fusion of endosomes with lysosomes and delaying vacuole acidification in macrophages [49]. For the design of a vaccine it is important to consider the balance between T helper cell type 1 (Th1, cellular immune response) and Th2 (humoral immune response) cells that are necessary for the pathogen clearance [7]. In Bcc
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vaccine design, the type of response necessary to clear the pathogen remains unknown. Moreover, CF patients show an altered immune phenotype, with a prevalence of Th2 responses. Th17 cells are another CD4+ T helper lineage that is involved in host defense of mucosal surfaces, in particular in the lungs, but which induces strong tissue damage [7]. Modulation of the Th17 response could be an interesting opportunity for Bcc vaccine development. Indeed, these three different types of cell-mediated immunity are necessary to obtain efficient protection against many different cellular and intracellular pathogens [50]. Updates on Vaccines against B. cenocepacia No effective vaccines to prevent B. cenocepacia infections are available. However, a great deal of effort has been put into the development of a vaccine against B. cenocepacia, and many studies have reported partial immunoprotection. Unfortunately, until now none has moved to clinical trials. The different methods of Burkholderia to survive intracellularly and to evade the immune system of the host add further complications in the production of an effective vaccine. The facultative intracellular life of B. cenocepacia and its complex interaction with the host represent a major challenge for the development of effective immunity. During the intracellular phase, Burkholderia antigens are likely to have limited visibility to the immune system. Therefore, although it is conceivable that antibodies play an important role in preventing bacterial entry into target cells, there is general consensus that full protection against intracellular pathogenic bacteria is achievable with a combination of humoral and cellular immune response. The statement is supported by the observation that well-balanced Th1/Th2 responses induced in CD-1 mice by vaccination with adjuvanted OmpW or OmpA resulted in effective reduction of B. cenocepacia burden in the lungs, that was distinct from the Th2-based immunity induced by OmpA alone [8,51], because to tackle the pathogen it is necessary to stimulate both humoral and cellular responses [5]. Table 2 and Figure 1 present an overview of antigens evaluated so far for the development of B. cenocepacia vaccines. Although there are some examples of live attenuated vaccines for related species such as Burkholderia mallei and Burkholderia pseudomallei, only one was recently described against Bcc (TonB), and this induced a strong Th1 response and was effective in clearing bacteria from mice [6] (Table 2). These results and the previous report on B. mallei [54] highlight the possibility to produce vaccine candidates with cross-protection among different Burkholderia spp., as in the case of FliC that was identified by protein microarray screening [10,55] (Table 2). Similar results were achieved using immunoproteomics screening of patient serum that discovered, among others, four immunogenic proteins common to both Burkholderia multivorans and B. cenocepacia strains: GroEL, 38 kDa porin, DNA-directed RNA polymerase (RNAP), and
Table 2. Overview of Antigens for the Development of B. cenocepacia Vaccines
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Antigen
Features
Refs
TonB
Membrane protein that interacts with outer-membrane receptors for iron uptake. tonB deletion affects B. cenocepacia survival during macrophage uptake, and mutants have an attenuated phenotype that is associated with reduced lethality in an in vivo mouse model
[6]
FliC
Flagellar conserved epitope between Burkholderia species that cause melioidosis and strains associated with cepacia syndrome in CF patients
[10]
Linocin
Well conserved in Bcc species; involved in the encapsulation of enzymes and in attachment to lung epithelial cells
[8]
OmpW
Outer-membrane protein found in many Gram-negative bacteria; involved in epithelial cell adhesion
[8]
OmpA
Outer-membrane protein associated with adhesion and invasion of host cells, induction of cell death, antimicrobial resistance, and immune evasion
[51–53]
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EF-Tu. This suggests the possibility of developing a multicomponent vaccine effective against both strains [56]. It has also been recently demonstrated that immunization of mice with OmpW protects against B. pseudomallei infection [57], and OmpW and linocin, identified by a novel proteomic approach, were also demonstrated to protect mice against B. cenocepacia and B. multivorans [8]. Both antigens elicited a serological response and were able to induce mixed Th1/Th2 responses, highlighting their adjuvant properties and promising potential as vaccines [8] (Table 2). Regarding the outer-membrane proteins (OMPS), another study demonstrated that a formulation of an OmpA-like polypeptide with an experimental noninflammatory mucosal adjuvant (NE) induced a high Bcc-specific serum and mucosal immune response in mice [51]. Recently, Sousa and coworkers showed that B. cenocepacia OmpA, that was previously found using a plasposon mutant library [58], is recognized by CF serum samples, showing that this protein can stimulate a humoral immune response and human neutrophil production, thus increasing the release of tumor necrosis factor (TNF)-α, elastase, myeloperoxidase, hydrogen peroxide, nitric oxide, and catalase [52]. All these data suggest that OmpA may also be a strong immunostimulant and a potential immunoprotectant against Bcc infections [52,53] (Table 2). Burkholderia polysaccharides could be considered to be attractive vaccine candidates because they stimulate the production of protective antibodies in humans [59]. Moreover, glycoconjugate vaccines induce an antibody response through a T cell-dependent pathway [60]. One of the first examples is the synthesis of the complex carbohydrate β-Kdo (3-deoxy-D-manno-oct-2ulosonic acid)-containing exopolysaccharide that is produced by B. pseudomallei and has been isolated from four Bcc spp. [61]. Another example is the synthesis of the tetrasaccharide outer core fragment of B. multivorans lipooligosaccharide [62]. Until now these synthetic oligosaccharides have not been tested against Bcc species as subunit vaccines. B. cenocepacia and Bcc spp. produce another polymer called cepacian, and studies on its structural conformation are ongoing to allow the design of a cepacian-based glycoconjugate vaccine against Bcc [23]. These results are promising, but unfortunately until now none of these glycoconjugates have been tested in animals. Furthermore, the polysaccharide conjugate CPS–CRM197 (capsular polysaccharide linked to the recombinant CRM197 diphtheria toxin mutant) together with the Hcp1 and TssM proteins was highly effective in mice against B. pseudomallei [63]. The most potent approach for vaccine design in the genomic era is 'reverse vaccinology' that relies on screening the entire bacterial genome for genes encoding putative antigens [64]. This approach could be implemented with the study of the 3D structure of the antigens of interest to allow the evaluation of individual antigenic domains [65]. Bcc vaccine design must consider that B. cenocepacia infections occur in nosocomial outbreaks or by environmental contamination, and for these reasons vaccine development should consider proteins relevant to human colonization that are expressed both by clinical and environmental species. Studies on the differential expression of OMP proteins of B. cenocepacia in four diverse growth conditions (soil, water, plants, and CF sputum) identified 72 proteins that are expressed in all these conditions. OMPs commonly expressed were resistance-nodulation-division (RND) efflux pumps, TonB siderophore receptors, and ABC transporters, and these could be considered promising targets for vaccine development [9]. Another study analyzed the gene expression profiles of 25 B. cenocepacia clinical isolates to identify factors associated with cepacia syndrome. Using a microarray-based approach, the authors found that bloodstream bacteria highly express the type III secretion system and the
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exopolysaccharide cepacian, whereas genes encoding the flagellar system were downregulated [66], thus suggesting other possible antigen candidates.
The Challenge for the Future In recent decades, advances in DNA sequencing technology and bioinformatics have produced an exponential growth of genome sequence data, thus accelerating the acquisition of knowledge on functional networks among bacterial genes and proteins, and providing new opportunities for vaccine development (Figure 1). The whole-genome sequences publicly available represent a valuable source of information in the search for novel vaccine candidates. Bioinformatic analysis of bacterial genomes can use a series of tools to rapidly identify proteins with the characteristics of vaccine candidates (extracellular localization, conservation among different strains, predicted high solubility, and the presence of B and T cell epitopes), thus bypassing the need to grow the pathogen (Figure 1). To date, 17 completed and fully annotated genomes of B. cenocepacia have been filed at the NCBI database site (https://www.ncbi.nlm.nih.gov/genome/microbes/). Unannotated chromosome sequences of additional three strains (VC2307, VC1254, and GIMC4560:Bcn122) and 279 unassembled isolate sequences are also available. The genetic repertoire of B. cenocepacia comprises two or three chromosomes plus in some cases an additional plasmid. Overall, the number of replicons varies between two and four, accounting for genome sizes ranging from 6.61 to 8.73 Mb that code for between 5759 and 7912 proteins. In addition to the already characterized virulence factors, the ensemble of predicted polypeptides can include, as in the case of strain J2315, N1700 hypothetical proteins, 41 of which are predicted to be extracellular or outer membrane-associated and N900 with an unpredicted cellular localization (data reported on the PsortB server website, https://db.psort.org/). This evaluation gives a flavor of how much the identification of novel antigens of B. cenocepacia could benefit from progress in characterizing the roles played by the ensemble of still functionally unknown proteins potentially encoded by the bacterium. Comparative genomics could also greatly contribute to identifying novel B. cenocepacia vaccine candidates. The ability to rapidly adapt to survive in human lungs, the increasing resistance to antibiotics, and the ability to form biofilms highlight Bcc and P. aeruginosa as problematic opportunistic pathogens in patients with CF. It has been reported that CF subjects infected with P. aeruginosa are vulnerable to secondary infections with B. cenocepacia complex strains, and that in many cases this co-colonization worsens patient prognosis [67,68]. Such considerations underline the medical need for novel therapeutics to target both bacterial infections in CF patients, and prompt investigations into common antigens that could in principle promote cross-protection. A whole-genome comparison of gene ortholog clusters between the B. cenocepacia J2315 and H111 and P. aeruginosa PAO1 is shown in Figure 2. Strain J2315 is a member of the highly transmissible epidemic ET12 lineage that is associated with severe infection and poor prognosis [69]. By contrast, H111 infection has not been associated with acute symptoms in CF patients [70]. Both J2315 and H111 strains share more than 2000 clusters of orthologs with PAO1. This overview suggests that the identification of common antigens between B. cenocepacia and P. aeruginosa is in principle feasible and highly desirable for the development of novel vaccines and therapeutic antibodies. The challenge of antigen diversity and variability can be overcome by the inclusion of multiple antigens. Multicomponent vaccines have the potential to induce antibodies that could interfere with key steps in virulence and mitigate the risk of immunoselection mechanisms. In addition to the in silico identification of potential surface-exposed proteins, other approaches for antigen discovery are suitable, including direct determination of the bacterial surface proteome. Using this strategy, Shinoy and colleagues [56] were able to identify a set of immunogenic proteins that are expressed by B. cenocepacia during infection. 8
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Figure 2. Distribution of Clusters of Protein Orthologs in Burkholderia cenocepacia Strains J2315 and H111, and Pseudomonas aeruginosa PAO1. Data were generated using the OrthoVenn 2 web server (https://orthovenn2. bioinfotoolkits.net/home) using predicted proteomes derived from complete genome sequences of strains available at the NCBI database.
The possibility to rapidly identify a large number of potential vaccine candidates can represent a challenge for the evaluation of their antigenicity because this would require the cloning and expression of hundreds of recombinant antigens following by immunization screening. For this reason, high-throughput methods for functional screening such as parallel immunoreactivity (IR)-ELISA [71], protein microarrays [72], bacterial transcriptome analysis [73], and in vivo induced antigen technology [74] represent valuable tools that can inform the process of candidate prioritization for costly and labor-intensive testing in animal models. Recent advances in mass spectrometry, cryo-electron microscopy coupled with NMR, crystallography, and the isolation and production of monoclonal antibodies are completely changing the landscape and opening the new era of 'structural vaccinology' in which antigens can be made more stable and more immunogenic by rational design. This approach proceeds mainly through the isolation of antibodies with functional activity that allow the mapping of most protective epitopes and the design of ideal vaccine antigens that are stable, highly immunogenic, and cross-protective. An example of such an elegant approach was the design of chimeric fHbp antigens (factor H binding protein of Meningococcus) by Scarselli et al. [75] in which the most important epitopes of the three main fHbp variants were combined into a new fHbp chimeric protein that was able to induce high levels of cross-bactericidal antibodies. Another example concerns the redesign of the F protein of RSV (respiratory syncytial virus). The recombinant F protein is highly unstable and changes conformation very quickly from the prefusion form (as in the viral capsid) to an open, postfusion, conformation that is adopted during fusion between the viral and eukaryotic membranes. The resolution of the 3D structures of the pre- and postfusion F proteins allowed the identification of two key residues that, when changed to cysteine, become coupled through a disulfide bond and stabilize the F protein in a
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prefusion form that is able to induce high levels of neutralizing antibodies that were never achieved before [76].
Concluding Remarks and Future Perspectives The success of these new technologies could open the way to the identification and modeling of Burkholderia antigens selected either by reverse vaccinology or through the isolation of monoclonal antibodies derived from immunized animals or isolated from infected patients that recognize key antigens (see Outstanding Questions). The new antigens could then be overexpressed in outer-membrane vesicles, which are known to act as an ideal delivery system for bacterial antigens, or be expressed in nanoparticles or used in combination with new adjuvants. Advances in understanding adaptive and innate immune mechanisms, the discovery of TLRs and their natural agonists, recognition of the importance of the way antigens in which are presented to the immune system, and of the potential role the microbiome can play in the immune response, are all making an important contribution to vaccine discovery and implementation. In conclusion, these novel technologies could allow the development of effective vaccines that have so far not been possible, and thus contribute to addressing the challenges of acute and chronic infections as well as antimicrobial resistance (see Outstanding Questions). Acknowledgments This work was supported by a grant from the Cystic Fibrosis Foundation (RICCAR17G0 to G.R.), a BlueSky research grant of
Outstanding Questions To what extent does interindividual heterogeneity in the CF airway microbiota contribute to reduced microbial susceptibility to antibiotic treatment? Why has the development of vaccines for CF pathogens not been successful so far? How will the employment of new technologies improve our knowledge about CF pathogens and lead to vaccine development? Will the development of a vaccine against multi-infections (e.g., B. cenocepacia and P. aeruginosa, or B. cepacia complex strains and B. pseudomallei) be possible? What advantages could it bring to patients? When should a vaccine against B. cenocepacia be administered to CF patients? Could it be used also for immunocompromised patients?
the University of Pavia (to S.B.), and funding from the Italian Ministry of Education, University, and Research (MIUR), Dipartimenti di Eccellenza Program (2018–2022), to the Department of Biology and Biotechnology 'L. Spallanzani', University of Pavia.
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Should vaccines be used in combination with antimicrobials? Will this strategy lead to the eradication of Burkholderia infections?
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Review
Vaccines to Overcome Antibiotic Resistance: The Challenge of Burkholderia cenocepacia Viola C. Scoffone,1,3 Giulia Barbieri,1,3 Silvia Buroni,1 Maria Scarselli,2 Mariagrazia Pizza,2 Rino Rappuoli,2 and Giovanna Riccardi1,* Cystic fibrosis (CF) patients are at particular risk of infection by microorganisms that are resistant to several antibiotics. About 3% of CF patients are colonized by Burkholderia cenocepacia, and this represents a major threat because of its intrinsic high level of drug resistance and the lack of a safe and effective treatment protocol. The development of anti-Burkholderia vaccines is a valuable and complementary approach, but only a few studies have been reported to date. In this review we discuss recent advances in the vaccine field and how new technologies, including structural reverse vaccinology, could drive the design of an effective vaccine against B. cenocepacia for use in preventive and therapeutic applications.
Highlights The management of infections caused by multidrug-resistant bacteria is particularly difficult in CF patients, where the complex lung environment in which many pathogens interact complicates testing antibiotic efficacy, leading to increased morbidity. Burkholderia cenocepacia represents a major threat to CF patients because of its intrinsic high level of drug resistance and the lack of a safe and effective treatment protocol, as well as lack of interest by the pharmaceutical industry and scientific journals given the small number of people suffering from this infection.
Antibiotic Resistance and Its Impact on CF Bacterial multidrug resistance is a major challenge of our time: as recently described by the World Health Organization, it represents a global priority, and for some dangerous pathogens a standard eradication protocol is still lacking (https://www.who.int/antimicrobial-resistance/ en/). In recent years many scientists have focused their research on CF patients who are at particular risk of infection by microorganisms resistant to several antibiotics [1]. CF is caused by deficiency in the CTFR (cystic fibrosis transmembrane conductance regulator) ion channel, which leads to the accumulation of dehydrated mucus at the level of different organs, particularly in the lungs, leading to the local proliferation of multiple opportunistic pathogens [2]. The definition and detection of antibiotic resistance is particularly challenging in CF because of the difficulty of recreating the complex CF lung environment where many pathogens interact, and of testing the efficacy of antibiotics in these conditions [1]. This complicates the management of infections and significantly contributes to reduced survival in individuals with CF. One opportunistic pathogen that is particularly difficult to treat is Burkholderia cenocepacia: this represents a major threat because of its intrinsic high level of resistance to drugs currently used in therapy and because of the lack of a safe and effective treatment protocol [3]. Because epidemiological data show that only a small percentage of CF patients (~3%) in the USA suffer from this infection, there has been little investment in the identification of new drugs [4]. In summary, B. cenocepacia is a deadly infectious agent that principally affects people with a rare genetic disease, and there has been limited interest in this opportunistic pathogen from researchers, the pharmaceutical industry, and scientific journals. Diffusion of information regarding this infection is crucial for helping the CF community.
1
Vaccine development represents a valuable and complementary approach in the battle against bacterial infections affecting CF patients. However, there have been only a few studies on the development of vaccines against B. cenocepacia [5–10]. In this review we discuss new technological advances in the vaccine field (including reverse vaccinology) that could drive the design of vaccines against B. cenocepacia for use as preventive and therapeutic agents. We also focus on monoclonal antibodies, a new strategy that could be applied to a poorly studied bac-
*Correspondence: [email protected] (G. Riccardi).
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A complementary approach might be the development of vaccines, supported by new advanced technologies in the field (including structural reverse vaccinology), for use as preventive and therapeutic agents.
Department of Biology and Biotechnology 'Lazzaro Spallanzani', University of Pavia, Pavia, Italy 2 GlaxoSmithKline, Siena, Italy 3 These authors contributed equally
https://doi.org/10.1016/j.tim.2019.12.005 © 2019 Elsevier Ltd. All rights reserved.
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terium such as B. cenocepacia. By dissecting the different aspects of B. cenocepacia pathogenesis, antimicrobial resistance, and potential preventive and therapeutic measures against these infections, we aim to raise attention to a deadly pathogen that is a major threat to many CF patients.
Lung Infections in CF Patients Microbial Communities in the CF Lung Environment CF patients suffer from chronic lung infections caused by complex polymicrobial communities that are responsible for inflammation and a progressive decline in lung function (Figure 1, Key Figure). Culture-independent, next-generation sequencing-based studies revealed that known CF pathogens (Pseudomonas aeruginosa, methicillin-resistant Staphylococcus aureus, and multiple species of Haemophilus and Burkholderia cepacia complex) coexist with additional members of the CF lung community – such as Stenotrophomonas maltophilia, Achromobacter spp., nontuberculous mycobacteria (Mycobacterium abscessus complex, Mycobacterium avium complex), the Streptococcus milleri group, and different anaerobes
Key Figure
Overview of the Key Points Described in the Review
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Figure 1. Cystic fibrosis (CF) patients are usually colonized by many bacteria. Of CF patients, 3% are colonized by Burkholderia cenocepacia (in red), for which only a few antibiotics are still effective. The lack of therapies is made worse by the intrinsic resistance of Burkholderia spp. to many antimicrobial compounds via different mechanisms. Putative antigens (mainly with extracellular localization) for vaccine development have already been identified, and the use of new technologies will contribute to the discovery of candidate vaccines with desired characteristics (created with BioRender.com). Abbreviations: EPS, exopolysaccharide; IR-ELISA, immunoreactive ELISA; LPS, lipopolysaccharide; MRSA, methicillin-resistant Staphylococcus aureus; RND, resistance-nodulation-division transporters.
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Box 1. Sources of Infections The airway microbiota is established early in life. The upper airways (nasal and oropharyngeal cavities) are initially colonized by a rich pool of bacteria that are then selected based on their ability to adapt to the new environmental niche. According to the current model, the rich upper airways act as reservoirs of bacteria that can then colonize the lower airway compartments [77]. In this context, the oral cavity represents the main source of bacteria of the lung microbiota. The throat microbiota in healthy and CF individuals is dominated by few genera, including Streptococcus, Veillonella, and Prevotella [16,78]. Interestingly, oral streptococci play a key role in influencing CF community dynamics. Different streptococcal species (Streptococcus milleri group, Streptococcus oralis, Streptococcus mitis, Streptococcus gordonii, and Streptococcus sanguinis) were reported to modulate the pathogenicity of P. aeruginosa [79,80] and to influence the composition of the microbial community by inhibiting P. aeruginosa colonization when streptococci are pioneer colonizers [80]. This observation acquires important relevance when we consider that Streptococcus is one of the most abundant genera in the airway microbiome of CF infants [13]. Although throat and lung show closely related microbiota, the nasal cavity is colonized by a different microbial community [16] that, in healthy adults, is dominated by the genera Propionibacterium, Moraxella, Corynebacterium, or Staphylococcus. CF patients were reported to display a distinct nasopharyngeal bacterial microbiota characterized by an increased abundance of Staphylococcus aureus [81,82]. This CF nasal microbiota pattern is acquired early in life and probably reflects the altered environment of the nasal cavity of CF patients which favors colonization by Staphylococcus while preventing the adaptation and growth of normal commensals. The nasal cavity can therefore represent the origin of S. aureus colonization of the lower airways that is very common in CF patients, especially during infancy. Lung microbial communities are also strongly influenced by the gut microbiota. A high degree of concordance between the gut and respiratory microbial communities was reported in CF infants, and a large number of genera were present in the gut before colonization of the respiratory tract [83]. Finally, some bacterial species that are part of the CF lung microbiota can be acquired from patient-to-patient transmission or from the environment. This is the case of P. aeruginosa and B. cepacia complex bacteria that can be acquired from environmental reservoirs and then spread among patients [84].
such as Prevotella, Rothia, and Veillonella – that could contribute to the disease [11]. The different sources of infections are reported in Box 1. The CF airway microbiota shows a significant interindividual heterogeneity in composition and diversity [12]. During the first 2 years of life, the airway microbial community develops gradually and is enriched in the genera Streptococcus and Haemophilus [13]. Bacterial diversity is greatest in early life until adolescence and then declines with increasing age and disease progression [12,14,15]. This reduction in diversity is generally associated with an increase in the prevalence and dominance of P. aeruginosa [12,16], an extremely virulent, Gram-negative bacterium whose higher abundance was historically considered to be the cause of the onset of pulmonary exacerbations [17]. However, longitudinal studies reported no significant relationship between P. aeruginosa levels and the onset of exacerbations, and showed an unexpected stability of the CF lung microbiota composition [13,18]. In addition to P. aeruginosa, Burkholderia cepacia complex (Bcc) spp. are abundant in the airway communities of CF adults [16,19]. Impact of Antibiotic Treatments The observation that frequent exposure to multiple antibiotics has only a transient impact on the airway microbiota of CF patients highlights the ability of these microbial communities to tolerate antibiotic treatment or to acquire antibiotic resistance. Tolerance to antibiotic can be due to the ability of pathogens to grow in biofilms (P. aeruginosa), to the development of persister cells, and to the establishment of complex interspecies interactions that limit the efficacy of antibiotic treatments [20]. The complexity of these interactions and the interindividual heterogeneity of the microbial communities colonizing the airways of CF patients represent a challenge for the efficacy of therapies, highlighting the need to develop new preventive and therapeutic strategies.
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The Case of B. cenocepacia Why Is B. cenocepacia Difficult to Treat and Eradicate? B. cenocepacia is one of the most clinically relevant members of the Bcc, a group of 24 closely related bacterial species [21], described for the first time by Walter Burkholder in 1949. B. cenocepacia is a ubiquitous nonglucose fermenter aerobic Gram-negative bacterium that is commonly isolated from water, soil, and from sputum of CF patients. The incidence of B. cenocepacia infections in CF patients is only 3%, but this infection leads to a 2.5-fold reduction in their life expectancy relative to noncolonized patients [22]. Indeed, infection can lead to the 'cepacia syndrome', a fatal necrotizing pneumonia characterized by fever and septicemia. CF patients infected by B. cenocepacia have a worse decline compared with patients infected by other CF pathogens such as P. aeruginosa. Moreover, Bcc contamination of medical devices, disinfectants, and pharmaceutical formulations leads to outbreaks among hospitalized non-CF patients such as those undergoing chemotherapy and immunosuppressed individuals. The numerous virulence factors of B. cenocepacia play an important role in its pathogenicity (Table 1). However, this bacterium is extremely difficult to treat and to eradicate because of its high level of antibiotic resistance caused by reduced of cell-wall permeability, increased activity of efflux pumps, mutations in antibiotic target genes, and enzymatic modification or inactivation of the drug (Figure 1). It is noteworthy that the ability to produce biofilm also greatly contributes to antibiotic resistance [3]. Although lung transplantation is an established strategy for end-stage CF patients to improve survival and quality of life, B. cenocepacia infections are reported as a contraindication leading to significantly worse outcome, and 1 year and 5 year survival rates of infected patients are 68% and 42.6%, respectively, versus 93.6% and 73.7% for B. cenocepacia-negative patients [28]. Therapeutic Options for Burkholderia Infections in CF Patients All Burkholderia features described above make it impossible to establish a standard treatment strategy to eradicate chronic infections [29]. The choice of effective antimicrobials is severely limited by the ability of Burkholderia spp. to adapt to adverse conditions including the presence of antibiotics. Indeed, there have been only a few reports of successful eradication treatment in recent years: in one study a child with CF underwent a combination of intravenous (IV) and nebulized antibiotics plus sinus surgery [30]. Garcia and collaborators instead used an intensive
Table 1. B. cenocepacia Virulence Factors
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Virulence factor
Features
Refs
Exopolysaccharide (cepacian)
Phagocytosis and reactive oxygen species (ROS) production interference
[23]
Biofilm formation
Enhanced in the presence of neutrophil-like dHL60 cells; bacterial protection from recognition by the immune system
[24]
Lipopolysaccharide
Infection establishment; neutrophil respiratory burst response and stimulation of the production of proinflammatory cytokines
[23,25]
Secretion systems
T2SS: zinc metalloprotease secretion T4SS-1 and T3SS: role in intracellular survival T5SS: useful for bacterial adhesion T6SS: affects the actin cytoskeleton of macrophages; activates the inflammasome and enhances the activity of caspase-1
[23]
Siderophores
Ornibactin, pyochelin, cepabactin, and cepaciachelin: iron chelation and uptake during infection
[26]
Flagellin
Infection establishment
[27]
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combination of IV, inhaled, and oral antibiotic therapies based on in vitro sensitivities [31]. A 2 week treatment with IV tobramycin, ceftazidime, and temocillin was used, followed by 3 months of inhaled tobramycin, to eradicate Burkholderia infections from two children in the UK [32]. However, the duration of therapy, the use of single or multiple therapy, and the lack of correlation between in vitro and in vivo susceptibility data are still under debate [33]. In addition to the drugs mentioned above (Figure 1), new compounds are in clinical trials, such as the inhaled alginate oligosaccharide (OligoG) whose potential for use in biomedical applications to prevent infections or biofilms has been recently reported [34]. Moreover, the challenge in finding a cure for Burkholderia infections led to the proposal of alternative approaches including (i) molecules already used for other diseases, such as immunosuppressors and corticosteroids, which was successful in two patients [35]; interferon-γ, which until now has been tested only ex vivo [36]; and thiosulfoacid S-esters [37], cysteamine [38], and imidazoles [39] whose activity has been assessed in vitro. (ii) Natural products with antimicrobial activity such as plant nanoparticles [40] that were tested in vitro; fish oils [41], which showed activity in a Galleria mellonella infection model; and glycopolymers [42], that were successfully used against Bcc CF clinical isolates. (iii) Other methods based on the use of phage therapy [43] or antimicrobial peptides that can block B. cenocepacia biofilm formation [44], and quorumsensing inhibitors to attenuate virulence [45]. In this scenario, the development of new therapeutic strategies against this bacterium is extremely important in order to improve the life expectancy of CF patients. Vaccines are the most effective weapons to fight the global rise of antimicrobial resistance because they do not induce selective pressure on the environment and they do not contribute to antimicrobial resistance.
Vaccines against B. cenocepacia Immune Response The characterization of the immune response to B. cenocepacia is extremely complex: CF patients have an abnormal immune regulation because bacteria are embedded in the mucin layer or in the intracellular environment, and this can interfere with the host immune response. The airway epithelium is overlaid by high molecular weight glycoproteins (mucins) that provide a physical barrier. In normal conditions, bacteria bind to the mucins and are cleared by the mucociliary activity. In CF patients, impaired mucociliary clearance leads to the persistence of bacteria in the airway lumen and, consequently, to their access to the epithelial cells. Different surface structures are involved in the interaction between B. cenocepacia and the host lung epithelium: flagella, pili, and adhesins [46]. Furthermore, bacterial lipopolysaccharide (LPS), flagella, pili, and lipopeptides are recognized by the innate immune Toll-like receptors (TLRs) that induce the production of proinflammatory cytokines [47]. B. cenocepacia can survive intracellularly in both airway epithelial cells and macrophages in order to evade host defenses and to establish chronic infections. Flagella and lipases promote invasion of lung epithelial cells, while intracellular survival and replication seem to require other factors such as the type IV secretion system [48]. During cell invasion, bacteria persist in the autophagosomes and replicate in the endoplasmic reticulum, showing that B. cenocepacia is able to subvert the normal endosomal pathway, thus preventing the fusion of endosomes with lysosomes and delaying vacuole acidification in macrophages [49]. For the design of a vaccine it is important to consider the balance between T helper cell type 1 (Th1, cellular immune response) and Th2 (humoral immune response) cells that are necessary for the pathogen clearance [7]. In Bcc
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vaccine design, the type of response necessary to clear the pathogen remains unknown. Moreover, CF patients show an altered immune phenotype, with a prevalence of Th2 responses. Th17 cells are another CD4+ T helper lineage that is involved in host defense of mucosal surfaces, in particular in the lungs, but which induces strong tissue damage [7]. Modulation of the Th17 response could be an interesting opportunity for Bcc vaccine development. Indeed, these three different types of cell-mediated immunity are necessary to obtain efficient protection against many different cellular and intracellular pathogens [50]. Updates on Vaccines against B. cenocepacia No effective vaccines to prevent B. cenocepacia infections are available. However, a great deal of effort has been put into the development of a vaccine against B. cenocepacia, and many studies have reported partial immunoprotection. Unfortunately, until now none has moved to clinical trials. The different methods of Burkholderia to survive intracellularly and to evade the immune system of the host add further complications in the production of an effective vaccine. The facultative intracellular life of B. cenocepacia and its complex interaction with the host represent a major challenge for the development of effective immunity. During the intracellular phase, Burkholderia antigens are likely to have limited visibility to the immune system. Therefore, although it is conceivable that antibodies play an important role in preventing bacterial entry into target cells, there is general consensus that full protection against intracellular pathogenic bacteria is achievable with a combination of humoral and cellular immune response. The statement is supported by the observation that well-balanced Th1/Th2 responses induced in CD-1 mice by vaccination with adjuvanted OmpW or OmpA resulted in effective reduction of B. cenocepacia burden in the lungs, that was distinct from the Th2-based immunity induced by OmpA alone [8,51], because to tackle the pathogen it is necessary to stimulate both humoral and cellular responses [5]. Table 2 and Figure 1 present an overview of antigens evaluated so far for the development of B. cenocepacia vaccines. Although there are some examples of live attenuated vaccines for related species such as Burkholderia mallei and Burkholderia pseudomallei, only one was recently described against Bcc (TonB), and this induced a strong Th1 response and was effective in clearing bacteria from mice [6] (Table 2). These results and the previous report on B. mallei [54] highlight the possibility to produce vaccine candidates with cross-protection among different Burkholderia spp., as in the case of FliC that was identified by protein microarray screening [10,55] (Table 2). Similar results were achieved using immunoproteomics screening of patient serum that discovered, among others, four immunogenic proteins common to both Burkholderia multivorans and B. cenocepacia strains: GroEL, 38 kDa porin, DNA-directed RNA polymerase (RNAP), and
Table 2. Overview of Antigens for the Development of B. cenocepacia Vaccines
6
Antigen
Features
Refs
TonB
Membrane protein that interacts with outer-membrane receptors for iron uptake. tonB deletion affects B. cenocepacia survival during macrophage uptake, and mutants have an attenuated phenotype that is associated with reduced lethality in an in vivo mouse model
[6]
FliC
Flagellar conserved epitope between Burkholderia species that cause melioidosis and strains associated with cepacia syndrome in CF patients
[10]
Linocin
Well conserved in Bcc species; involved in the encapsulation of enzymes and in attachment to lung epithelial cells
[8]
OmpW
Outer-membrane protein found in many Gram-negative bacteria; involved in epithelial cell adhesion
[8]
OmpA
Outer-membrane protein associated with adhesion and invasion of host cells, induction of cell death, antimicrobial resistance, and immune evasion
[51–53]
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EF-Tu. This suggests the possibility of developing a multicomponent vaccine effective against both strains [56]. It has also been recently demonstrated that immunization of mice with OmpW protects against B. pseudomallei infection [57], and OmpW and linocin, identified by a novel proteomic approach, were also demonstrated to protect mice against B. cenocepacia and B. multivorans [8]. Both antigens elicited a serological response and were able to induce mixed Th1/Th2 responses, highlighting their adjuvant properties and promising potential as vaccines [8] (Table 2). Regarding the outer-membrane proteins (OMPS), another study demonstrated that a formulation of an OmpA-like polypeptide with an experimental noninflammatory mucosal adjuvant (NE) induced a high Bcc-specific serum and mucosal immune response in mice [51]. Recently, Sousa and coworkers showed that B. cenocepacia OmpA, that was previously found using a plasposon mutant library [58], is recognized by CF serum samples, showing that this protein can stimulate a humoral immune response and human neutrophil production, thus increasing the release of tumor necrosis factor (TNF)-α, elastase, myeloperoxidase, hydrogen peroxide, nitric oxide, and catalase [52]. All these data suggest that OmpA may also be a strong immunostimulant and a potential immunoprotectant against Bcc infections [52,53] (Table 2). Burkholderia polysaccharides could be considered to be attractive vaccine candidates because they stimulate the production of protective antibodies in humans [59]. Moreover, glycoconjugate vaccines induce an antibody response through a T cell-dependent pathway [60]. One of the first examples is the synthesis of the complex carbohydrate β-Kdo (3-deoxy-D-manno-oct-2ulosonic acid)-containing exopolysaccharide that is produced by B. pseudomallei and has been isolated from four Bcc spp. [61]. Another example is the synthesis of the tetrasaccharide outer core fragment of B. multivorans lipooligosaccharide [62]. Until now these synthetic oligosaccharides have not been tested against Bcc species as subunit vaccines. B. cenocepacia and Bcc spp. produce another polymer called cepacian, and studies on its structural conformation are ongoing to allow the design of a cepacian-based glycoconjugate vaccine against Bcc [23]. These results are promising, but unfortunately until now none of these glycoconjugates have been tested in animals. Furthermore, the polysaccharide conjugate CPS–CRM197 (capsular polysaccharide linked to the recombinant CRM197 diphtheria toxin mutant) together with the Hcp1 and TssM proteins was highly effective in mice against B. pseudomallei [63]. The most potent approach for vaccine design in the genomic era is 'reverse vaccinology' that relies on screening the entire bacterial genome for genes encoding putative antigens [64]. This approach could be implemented with the study of the 3D structure of the antigens of interest to allow the evaluation of individual antigenic domains [65]. Bcc vaccine design must consider that B. cenocepacia infections occur in nosocomial outbreaks or by environmental contamination, and for these reasons vaccine development should consider proteins relevant to human colonization that are expressed both by clinical and environmental species. Studies on the differential expression of OMP proteins of B. cenocepacia in four diverse growth conditions (soil, water, plants, and CF sputum) identified 72 proteins that are expressed in all these conditions. OMPs commonly expressed were resistance-nodulation-division (RND) efflux pumps, TonB siderophore receptors, and ABC transporters, and these could be considered promising targets for vaccine development [9]. Another study analyzed the gene expression profiles of 25 B. cenocepacia clinical isolates to identify factors associated with cepacia syndrome. Using a microarray-based approach, the authors found that bloodstream bacteria highly express the type III secretion system and the
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exopolysaccharide cepacian, whereas genes encoding the flagellar system were downregulated [66], thus suggesting other possible antigen candidates.
The Challenge for the Future In recent decades, advances in DNA sequencing technology and bioinformatics have produced an exponential growth of genome sequence data, thus accelerating the acquisition of knowledge on functional networks among bacterial genes and proteins, and providing new opportunities for vaccine development (Figure 1). The whole-genome sequences publicly available represent a valuable source of information in the search for novel vaccine candidates. Bioinformatic analysis of bacterial genomes can use a series of tools to rapidly identify proteins with the characteristics of vaccine candidates (extracellular localization, conservation among different strains, predicted high solubility, and the presence of B and T cell epitopes), thus bypassing the need to grow the pathogen (Figure 1). To date, 17 completed and fully annotated genomes of B. cenocepacia have been filed at the NCBI database site (https://www.ncbi.nlm.nih.gov/genome/microbes/). Unannotated chromosome sequences of additional three strains (VC2307, VC1254, and GIMC4560:Bcn122) and 279 unassembled isolate sequences are also available. The genetic repertoire of B. cenocepacia comprises two or three chromosomes plus in some cases an additional plasmid. Overall, the number of replicons varies between two and four, accounting for genome sizes ranging from 6.61 to 8.73 Mb that code for between 5759 and 7912 proteins. In addition to the already characterized virulence factors, the ensemble of predicted polypeptides can include, as in the case of strain J2315, N1700 hypothetical proteins, 41 of which are predicted to be extracellular or outer membrane-associated and N900 with an unpredicted cellular localization (data reported on the PsortB server website, https://db.psort.org/). This evaluation gives a flavor of how much the identification of novel antigens of B. cenocepacia could benefit from progress in characterizing the roles played by the ensemble of still functionally unknown proteins potentially encoded by the bacterium. Comparative genomics could also greatly contribute to identifying novel B. cenocepacia vaccine candidates. The ability to rapidly adapt to survive in human lungs, the increasing resistance to antibiotics, and the ability to form biofilms highlight Bcc and P. aeruginosa as problematic opportunistic pathogens in patients with CF. It has been reported that CF subjects infected with P. aeruginosa are vulnerable to secondary infections with B. cenocepacia complex strains, and that in many cases this co-colonization worsens patient prognosis [67,68]. Such considerations underline the medical need for novel therapeutics to target both bacterial infections in CF patients, and prompt investigations into common antigens that could in principle promote cross-protection. A whole-genome comparison of gene ortholog clusters between the B. cenocepacia J2315 and H111 and P. aeruginosa PAO1 is shown in Figure 2. Strain J2315 is a member of the highly transmissible epidemic ET12 lineage that is associated with severe infection and poor prognosis [69]. By contrast, H111 infection has not been associated with acute symptoms in CF patients [70]. Both J2315 and H111 strains share more than 2000 clusters of orthologs with PAO1. This overview suggests that the identification of common antigens between B. cenocepacia and P. aeruginosa is in principle feasible and highly desirable for the development of novel vaccines and therapeutic antibodies. The challenge of antigen diversity and variability can be overcome by the inclusion of multiple antigens. Multicomponent vaccines have the potential to induce antibodies that could interfere with key steps in virulence and mitigate the risk of immunoselection mechanisms. In addition to the in silico identification of potential surface-exposed proteins, other approaches for antigen discovery are suitable, including direct determination of the bacterial surface proteome. Using this strategy, Shinoy and colleagues [56] were able to identify a set of immunogenic proteins that are expressed by B. cenocepacia during infection. 8
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Figure 2. Distribution of Clusters of Protein Orthologs in Burkholderia cenocepacia Strains J2315 and H111, and Pseudomonas aeruginosa PAO1. Data were generated using the OrthoVenn 2 web server (https://orthovenn2. bioinfotoolkits.net/home) using predicted proteomes derived from complete genome sequences of strains available at the NCBI database.
The possibility to rapidly identify a large number of potential vaccine candidates can represent a challenge for the evaluation of their antigenicity because this would require the cloning and expression of hundreds of recombinant antigens following by immunization screening. For this reason, high-throughput methods for functional screening such as parallel immunoreactivity (IR)-ELISA [71], protein microarrays [72], bacterial transcriptome analysis [73], and in vivo induced antigen technology [74] represent valuable tools that can inform the process of candidate prioritization for costly and labor-intensive testing in animal models. Recent advances in mass spectrometry, cryo-electron microscopy coupled with NMR, crystallography, and the isolation and production of monoclonal antibodies are completely changing the landscape and opening the new era of 'structural vaccinology' in which antigens can be made more stable and more immunogenic by rational design. This approach proceeds mainly through the isolation of antibodies with functional activity that allow the mapping of most protective epitopes and the design of ideal vaccine antigens that are stable, highly immunogenic, and cross-protective. An example of such an elegant approach was the design of chimeric fHbp antigens (factor H binding protein of Meningococcus) by Scarselli et al. [75] in which the most important epitopes of the three main fHbp variants were combined into a new fHbp chimeric protein that was able to induce high levels of cross-bactericidal antibodies. Another example concerns the redesign of the F protein of RSV (respiratory syncytial virus). The recombinant F protein is highly unstable and changes conformation very quickly from the prefusion form (as in the viral capsid) to an open, postfusion, conformation that is adopted during fusion between the viral and eukaryotic membranes. The resolution of the 3D structures of the pre- and postfusion F proteins allowed the identification of two key residues that, when changed to cysteine, become coupled through a disulfide bond and stabilize the F protein in a
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prefusion form that is able to induce high levels of neutralizing antibodies that were never achieved before [76].
Concluding Remarks and Future Perspectives The success of these new technologies could open the way to the identification and modeling of Burkholderia antigens selected either by reverse vaccinology or through the isolation of monoclonal antibodies derived from immunized animals or isolated from infected patients that recognize key antigens (see Outstanding Questions). The new antigens could then be overexpressed in outer-membrane vesicles, which are known to act as an ideal delivery system for bacterial antigens, or be expressed in nanoparticles or used in combination with new adjuvants. Advances in understanding adaptive and innate immune mechanisms, the discovery of TLRs and their natural agonists, recognition of the importance of the way antigens in which are presented to the immune system, and of the potential role the microbiome can play in the immune response, are all making an important contribution to vaccine discovery and implementation. In conclusion, these novel technologies could allow the development of effective vaccines that have so far not been possible, and thus contribute to addressing the challenges of acute and chronic infections as well as antimicrobial resistance (see Outstanding Questions). Acknowledgments This work was supported by a grant from the Cystic Fibrosis Foundation (RICCAR17G0 to G.R.), a BlueSky research grant of
Outstanding Questions To what extent does interindividual heterogeneity in the CF airway microbiota contribute to reduced microbial susceptibility to antibiotic treatment? Why has the development of vaccines for CF pathogens not been successful so far? How will the employment of new technologies improve our knowledge about CF pathogens and lead to vaccine development? Will the development of a vaccine against multi-infections (e.g., B. cenocepacia and P. aeruginosa, or B. cepacia complex strains and B. pseudomallei) be possible? What advantages could it bring to patients? When should a vaccine against B. cenocepacia be administered to CF patients? Could it be used also for immunocompromised patients?
the University of Pavia (to S.B.), and funding from the Italian Ministry of Education, University, and Research (MIUR), Dipartimenti di Eccellenza Program (2018–2022), to the Department of Biology and Biotechnology 'L. Spallanzani', University of Pavia.
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