The new first-line defense: the potential of nasopharyngeal colonization in vaccine strategies

Introduction

Nasopharyngeal commensal species

The upper respiratory tract is a host to many commensal bacterial species that create a complex community of microbes. These commensal species include a number of potentially pathogenic bacteria that usually colonize the nasopharynx without further progression to disease, but occasionally can spread from the nasopharynx to the lungs or blood to cause serious infections such as pneumonia, septicemia, and meningitis. Potential pathogens found in the nasopharynx include Streptococcus pneumoniae, Hemophilus influenzae, Neisseria meningitidis, Moraxella catarrhalis, and Staphylococcus aureus.¹ The nasopharynx provides a relatively stable environment in which the commensal flora can flourish and be transmitted to other hosts.^2,3 The duration of carriage varies for each species and also between the different strains of the same species, from weeks to months and likely to be years for some species. The dynamics of nasopharyngeal colonization by different species are delicately balanced, and disturbances to this ecology may allow pathogenic organisms to cause disease.⁴ Factors that affect this balance include the acquisition of different species, interspecies interactions, bacterial interactions with the host, and interference by environmental factors. For instance, nasopharyngeal colonization by S. pneumoniae protects against S. aureus carriage,⁵ and conversely reduction in S. pneumoniae carriage by vaccination could lead to increased carriage of S. aureus. Similarly, in animal models, bacterial species compete in establishing colonization.⁶

Colonization of the nasopharynx by bacteria can influence the host immune system even in the absence of overt disease. Epidemiologic studies have shown that the development of asthma^7–9 and chronic obstructive pulmonary disease^10,11 is related to the diversity of colonizing organisms in the nasal flora. In mice, lack of microbial colonization increases allergic airway inflammation,^12,13 and colonization with multiple species protects against airway inflammation.¹⁴ Importantly, nasopharyngeal colonization by bacterial pathogens can be an immunizing event, stimulating both humoral and cellular adaptive immune responses that protect against either re-colonization or subsequent invasive disease.^15–21 These observations suggest that novel vaccine strategies could harness the immunizing effects of nasopharyngeal colonization to prevent serious infections, and this is the subject addressed in this review with a particular focus on the potential of nasopharyngeal colonization for the prevention of S. pneumoniae and N. meningitidis infections, which are used as case studies that illustrate the benefits and potential drawbacks of this approach.

Colonization and existing vaccines for S. pneumoniae and N. meningitidis

S. pneumoniae

Colonization with S. pneumoniae is universal in the first few months of life, with between 50% and 90% of children aged under 2 years colonized at any one time,^22–27 sometimes with multiple strains.²⁸ Peak carriage rates occur at 3–5 years of age and then wane to ~10% in adult life.^6,29 Carriage prevalence depends on geographical location, and is generally higher in the developing world.^30–32 While initial colonization events may persist for up to 4 months, duration appears to shorten with increasing age to 2–4 weeks in adults.³³ The proportion of S. pneumoniaecolonization events associated with disease is low in healthy adults. However, as colonization is very common, S. pneumoniae is a leading cause of acute otitis media (OM), pneumonia, sepsis, and meningitis globally^34–36 causing an estimated 2,858,000 severe pneumonia episodes and 411,000 deaths annually worldwide in infants.³⁷ The use of vaccines targeting polysaccharide capsule antigen in children has reduced the overall incidence of pneumococcal disease.^38,39 However, the adult vaccine fails to protect against pneumonia,⁴⁰ a key cause of respiratory morbidity and mortality in elderly subjects with comorbidities. In addition, the existing vaccines have a high cost of manufacture and have major limitations in strain coverage, only protecting against between seven and 23 of the 93+ S. pneumoniae capsular serotypes. This restricted serotype coverage has led to the replacement of S. pneumoniae vaccine serotypes by non-vaccine serotypes as both colonizers of the nasopharynx and causes of disease.^31,41–43 Hence, there is a strong interest in alternative vaccine strategies that target all S. pneumoniae strains and could also prevent lung infection.

In addition to the profound effect on the relative prevalence of vaccine and non-vaccine S. pneumoniae serotypes, the introduction of the pneumococcal conjugate vaccine to infant immunization schedules has also disrupted the ecology of the nasopharyngeal flora in general.^44,45 Although the short- and long-term consequences of these changes are not yet clear, they still raise some potential concerns about the effects of vaccination. The complete eradication of all S. pneumoniae from the nasopharynx may remove the competition that S. pneumoniae exerts on other potentially pathogenic organisms, perhaps allowing their overgrowth. This could in turn lead to a greater incidence of disease caused by H. influenzae, N. meningitidis, S. aureus, or M. catarrhalis.¹ This disruption to the nasal flora will likely have implications on the incidence of disease and antibiotic strategies in the future.^5,46

N. meningitidis

N. meningitidis is a pathogenic member of the Neisseria family, several of which are common human nasopharyngeal commensal species.⁴⁷ N. meningitidis is a major cause of rapidly progressive meningitis and septicemia, which, although rare, often result in death or permanent disability. Nevertheless, N. meningitidis is an asymptomatic nasopharyngeal colonizer in ~10% of healthy individuals at any time.^48,49 The carriage rate is the lowest in young children and highest in young adults, with the UK rates of 3% of children under 4 years, 24%–37% of the age group of 15–24 years, and <10% in older age groups.^49,50 Similar to S. pneumoniae, carriage rates are higher in smokers and after viral respiratory tract infections.^48,51

Twelve different meningococcal serogroups have been defined based on capsular polysaccharide structure, of which A, B, C, W135, and Y are responsible for the majority of the disease. In addition, serogroup X has more recently been identified as the cause of sepsis and meningitis in Africa.⁵² Group A causes large-scale epidemics mainly in Africa but also in Asia, whereas the majority of cases of N. meningitidis disease in Europe and America are caused by serogroups B and C strains. As with S. pneumoniae, vaccines are currently polysaccharide based with newer vaccines utilizing protein conjugation to offer improved protection in children. In the UK, a conjugate vaccine to protect against serotype C has been in use since 1999 in infants, and also for teenagers and young adults. Unlike the serotype replacement seen with the introduction of the pneumococcal conjugate vaccine, the introduction of a vaccine for meningococcus C has not seen a rise in carriage or disease by meningococcus B. A conjugate vaccine targeting groups A, C, W135, and Y has been administered to adolescents since early 2016. Serogroup B has a capsule that is particularly poorly immunogenic, and the meningococcus B vaccine is based on outer membrane vesicles and protein antigens rather than capsular polysaccharide. This vaccine was added to the UK infant immunization schedule in 2015. Vaccine-related reduction in carriage may have contributed to herd immunity as was seen for S. pneumoniae.^53,54

Regulation by commensal species

The nasal flora is acquired shortly after birth and is influenced by the environment, including contact with other persons.⁵⁵ The competition between potentially pathogenic bacteria and commensal species in the nasopharynx contributes to the regulation of pathogenic species, which are also influenced by host responses. Environmental changes such as the season will influence the makeup of the nasal flora,⁵⁶ as will therapeutic interventions such as vaccination or antimicrobials. For example, children with pneumococcal OM treated with antibiotics, or those who were immunized with the pneumococcal conjugate vaccine, had a decrease in the prevalence of Streptococcaceae and Corynebacteriaceae commensal species in the nasopharynx.⁵⁷ Nasopharyngeal commensal species could prevent respiratory and invasive disease caused by pathogenic commensal species through a number of different mechanisms. These include the inhibition of colonization by potential pathogens by competition, either passively (occupying the same ecological niche) or actively (via direct growth inhibition or killing of competitor species). For example, in the gut, competition for nutrients causes a process referred to as colonization resistance, which is integral to controlling pathogenic bacteria such as enterohemorrhagic Escherichia coli, or Clostridium difficile,^58,59 and similar processes are likely in the nasopharynx. Commensal species can also produce antimicrobial peptides that directly affect pathogen growth or survival. For example, the poorly pathogenic commensal species, Streptococcus salivarius, produces bacteriocins that inhibit S. pneumoniae,^60,61 and in the gut enterohemorrhagic E. coli is inhibited by bacteriocins produced by other E. coli strains.⁶² On the skin, the commensal species, Staphylococcus epidermidis, produces antimicrobial proteins that prevent S. aureus growth.⁶³ Another mechanism that directly inhibits other bacteria species is the production of hydrogen peroxide (H₂O₂). S. pneumoniae is remarkably tolerant to H₂O₂ and although potential pathogens such as S. aureus and H. influenzae produce a catalase to neutralize H₂O_2, the concentrations produced by S. pneumoniae overwhelm these catalases without killing the S. pneumoniae itself.^46,64 Another strategy employed by S. pneumoniae is the production of neuraminidase, an enzyme that degrades H. influenzae cell-surface sialic acids, impairing the ability of H. influenzae to colonize the host.⁶⁵ Commensal nasopharyngeal flora can inhibit the growth of group A Streptococcus, although the mechanisms are not clear.⁶⁶ Finally, the impact of nasopharyngeal bacteria and viruses on other species can also be mediated via the modulation of the host’s immune response.⁴ For example, in a mouse model, initial colonization with H. influenzae stimulated an innate immune response via immune recognition of cell wall components that enhanced phagocytosis of S. pneumoniae and inhibited colonization.⁶⁷

Immunizing effect of colonization

Both human and animal data demonstrate that colonization is an immunizing event that prevents subsequent S. pneumoniae infection by both homologous and heterologous strains. Antibodies targeting capsular polysaccharides are detected in the serum of children following colonization,⁶⁸ although the strength of the immune response depends on the infecting serotype.^69,70 Exposure to a greater number of serotypes also enhances immune responses.⁷¹ In addition to anti-capsular antibodies, colonization in humans and in animal models induces antibodies to surface and intracellular S. pneumoniae protein antigens, many of which are protective.^6,72 Unlike anti-capsular responses, anti-protein responses are rapidly detectable in the first year of life.^68,73,74 However, an epidemiological study in infants did not find evidence that anti-protein antibodies protected against subsequent colonization.⁷⁵ Colonization of adult volunteers with serotype 23F or 6B strains of S. pneumoniae led to the production of serum IgG to the protein antigens PspC and PspA and salivary IgA to PspA⁷⁶ and preexisting serum anti-PspA IgG levels correlating with protection against experimental colonization.⁷⁷ In another experimental human challenge model, nasopharyngeal colonization with a serotype 6B strain of S. pneumoniae was established in healthy adults. Rechallenge failed to result in a second carriage event by the same strain, with protection persisting for up to 1 year.²¹ In these challenge studies, colonization resulted in cellular and humoral immune responses to S pneumoniae. Data from mouse colonization models indicate that nasopharyngeal colonization leads to Th17-cell responses^16,18,19 that enhance phagocyte recruitment to the nasopharynx,^18,78 and are critical for both the initial clearance of the colonizing strain and subsequent protection against recolonization. The impact of these phagocytic responses is enhanced by the effects of specific antibody via opsonization and agglutination of S. pneumoniae.⁷⁹ A murine model of group A Streptococcus nasopharyngeal infection has also been established.⁸⁰ In this model, rapid clearance of recolonization was also dependent on an antigen-specific Th17-cell response.⁸¹ These data raise the possibility that Th17-cell mechanisms may be broadly important in the control of bacterial colonization of the nasopharynx.

Human colonization also leads to serum antibody responses that are able to protect against sepsis caused by heterologous strains in mouse challenge models,⁷⁶ indicating that colonization-induced anti-protein responses are sufficient to enable protection. In addition to Th17-cell responses, the antibody response to colonization is also important for protection against S. pneumoniae lung infection in a mouse model.¹⁹ In the human experimental challenge model, colonization augmented S. pneumoniae-specific IL-17-secreting CD4+ T-cells in the human lung,²⁰ suggesting that there may also be a role for colonization-induced Th17 cells in limiting lung infection in humans.

Overall, these observations demonstrate that colonization of healthy humans induces a mucosal and systemic immune response that protects against further colonization (in humans) or sepsis (in mice), and support data from animal models show similar findings.^15,16,18,19 Importantly, intranasal administration of the 6B strain without successful colonization also augmented local mucosal serological responses,⁸² showing that exposure of the nasopharynx to the organism can stimulate an immunological response even if colonization was not detected.

Colonization of the upper airways with N. meningitidis is also an immunizing event that induces an antibody response persisting several weeks following acquisition.^83,84 Interestingly, childhood nasopharyngeal exposure to commensal strains of Neisseria increases antibody levels which is largely strain-specific but has some degree of cross-reactivity with N. meningitidis, and which perseveres for several months.^85–87

Overall, there is now an abundance of data indicating that nasopharyngeal colonization with potentially pathogenic bacteria elicits both humoral and cellular protective adaptive immune responses in humans and mouse models. These data support colonization as a novel alternative vaccine strategy to induce protection against bacterial pathogens.^88,89

Colonization as a vaccine strategy

There are two potential strategies by which colonization could be used to prevent disease. The first is through harnessing the regulatory effects of commensal species on colonization by potential pathogens through competition for resources, immune modulatory effects, the secretion of bacteriocins, or other direct inhibitory mechanisms. The second is by stimulating a protective adaptive immune response, which unlike the first strategy requires colonization by an organism with significant antigenic overlap to the target pathogen.

Prevention of colonization using commensal species

The delivery of nonpathogenic commensal species as “probiotics” has been investigated for the prevention of OM. In some cases, the impact on bacterial nasopharyngeal colonization has also been assessed.⁹⁰ These avirulent organisms were administered orally or via nasal spray, and the preliminary results suggest that they can reduce the incidence of upper respiratory infections.⁹¹ In a study of adults given an oral mixture of organisms (containing Lactobacillus rhamnosus GG, Bifidobacterium, Lactobacillus acidophilus, and Streptococcus thermophilus), there was a significant reduction in nasal colonization with potential pathogens, including S. aureus, S. pneumoniae, and β-hemolytic streptococci.⁹² Studies in children given milk supplemented with Lactobacillus rhamnosus GG tend to show a reduction in respiratory infections, including OM, but have produced mixed results in the impact on the carriage of pathogens such as S. pneumoniae and H. influenzae.^93,94 These studies delivered the commensal organisms to the gut where they might enhance general mucosal immunity through interactions with the gut-associated immune system.⁹²

Children who suffer from repeated episodes of OM are less likely to carry the oropharyngeal commensal alpha-hemolytic streptococci (AHS), whereas H. influenzae is more prevalent.⁹⁵ This observation perhaps suggests that AHS have inhibitory effect on H. influenzae growth, similar to the ability of Streptococcus oralis to inhibit S. pneumoniae growth.⁹⁶ This potential effect has been exploited by the oral administration of another AHS species S. salivarius, which was associated with a reduction in S. pyogenes infections in humans^97,98 and inhibition of S. pneumoniae infection in mouse models.^99,100 Furthermore, nasal spray administration of five strains of AHS from three species (Streptococcus sanguis, Streptococcus mitis, and S. oralis) reduced the incidence of recurrent OM and secretory OM in children.¹⁰¹ However, in another study, there was no significant change in the incidence of OM although there was a trend toward reduced carriage of H. influenzae.¹⁰² It is possible that in these studies, the administration of antibiotics prior to the bacterial nasal spray may have enabled stable colonization with AHS strains and therefore may make a positive result more likely.

Colonization to induce adaptive immunity

The remainder of this review focuses on using intranasal administration of bacteria as an immunizing event; these approaches are summarized in Table 1. This approach has a number of potential advantages to using conventional vaccines. A whole bacterial cell approach means that the immunological response is not restricted to selected antigens and will induce redundant responses to multiple antigens, improving cross-reactivity between strains and reducing the potential for naturally occurring vaccine escape mutations. In addition, the vaccine would be inexpensive to manufacture and would not need an adjuvant as antigens are presented in an immunostimulatory context of the whole bacterium. Nasal administration also offers the advantages over parenteral administration of higher safety levels, needleless delivery, and improved immunity at the mucosal surface which may be more likely to prevent respiratory tract infections. The key principles of colonization-induced immunity (Figure 1) are detailed in the following sections.

Table 1 Examples and potential mechanisms for inducing adaptive immunity to bacterial pathogens by nasopharyngeal colonization with live bacteria Abbreviations: L. lactis, Lactococcus lactis; N. lactamica, Neisseria lactamica; N. meningitidis, Neisseria meningitidis; S. pneumoniae, Streptococcus pneumoniae; S. pyogenes, Streptococcus pyogenes..

Figure 1 Summary of the principles employed in nasal vaccination strategies.

Cross-reactive protection between commensal and potentially pathogenic species

Colonization with a commensal species could potentially enhance the clearance of a closely related potential pathogen if there are shared antigens between the species. This has been explored for the closely related species Neisseria lactamica and N. meningitidis. N. lactamica expresses antigens similar to those expressed by N. meningitidis, and sera from mice immunized with N. lactamica enhance N. meningitidis killing,¹⁰³ and human carriage of N. lactamica results in a high titer of antibodies to N. meningitidis.¹⁰⁴ Outer membrane proteins and lipooligosaccharide structures common to both species are the major antigenic sources of cross-protection.¹⁰⁵ N. lactamica colonization has been studied as a vaccination strategy to prevent N. meningitidis disease. In one study of colonization of healthy volunteers, the mucosal and systemic antibody response against N. lactamica was cross-reactive against N. meningitidis. However, while these antibodies were opsonophagocytic in vitro, they had poor serum bactericidal effect. Furthermore, experimental N. lactamica colonization did not protect against subsequent natural N. meningitidis carriage acquisition.¹⁰⁴ In fact, there has been some evidence to suggest that N. lactamica even protects N. meningitidis during colonization by triggering antibody-independent responses that do not induce a memory response.¹⁰⁶ Nonetheless, the immunological response to colonization with N. lactamica could potentially protect against systemic infection with N. meningitidis, and further investigation of this strategy is ongoing. A similar strategy, in theory, could be applicable to other related pairs of pathogenic and nonpathogenic species, such as S. pneumoniae and S. mitis.

Attenuated pathogenic bacteria

Observations that natural mucosal exposure induces antibody and cellular immune responses to a range of bacterial antigens suggest that an alternative to current vaccine strategies could be the colonization of the nasopharynx with whole bacteria. This would reflect a more natural situation than subunit vaccines. To avoid the potential for causing active invasive infection, vaccination by artificial colonization of the nasopharynx would need to use attenuated strains unable to cause serious infection. This can now be achieved by targeted mutation of important virulence determinants, although these mutations could reduce the antigenicity of the attenuated strain. A critical aspect in the design of a live attenuated mucosal vaccine is achieving the balance between virulence attenuation for safety while retaining immunogenicity.

The use of attenuated S. pneumoniae as vaccines has been explored by several groups in animal models. In one example, genes encoding the capsule, Ply, and PspA were deleted rendering these strains avirulent yet still able to colonize and induce both systemic and mucosal antibodies that protected against disease in mice.¹⁰⁷ A similar approach was the SPY1 mutant strain, where the capsule, teichoic acids, and Ply were deleted from a D39 S. pneumoniae strain and used for intranasal immunization. This protected against colonization and invasive disease caused by heterologous strains of S. pneumoniae in a T-cell- and B-cell-dependent manner.^108,109 Further examples include the deletion of pep27 leading to an avirulent strain with reduced capsule expression which when used to immunize mice intranasally protected against colonization and systemic infection¹¹⁰ and double mutation of the signal recognition pathway component ftsY and the calcium/magnesium transporter caxP/mgtA which when administered to the nasopharynx induces heterologous protection against OM, pneumonia, and invasive disease in a CD4+ cell-dependent manner.¹¹¹ Another strategy for generating live vaccines is by creating auxotrophic organisms. In this way, key protective surface antigens such as PspA and the polysaccharide capsule for S. pneumoniae can be retained. For example, the deletion of the pabB gene creates an S. pneumoniae mutant strain auxotrophic for para-aminobenzoic acid and unable to replicate in the mammalian host. Systemic vaccination with this mutant was able to protect from homologous challenge in mouse models of sepsis and pneumonia.¹¹² The attenuated mutant approach has been investigated for other bacteria. For example, in S. pyogenes, the deletion of the SpeB protease creates a mutant that is impaired in tRNA modification and has the potential to be used in vaccination strategies.¹¹³ Mutation of gidA in Salmonella enterica serovar Typhimurium also lends itself to a novel vaccine strategy for this bacterium.¹¹⁴

A potentially significant problem is that mouse and human data indicate that a reduced duration of nasopharyngeal colonization by S. pneumoniae significantly weakens the induced adaptive immune response.^82,111,115 In mice, attenuated strains often have a reduced duration of colonization compared to wild-type bacteria,^107,111,115 and this may therefore affect the efficacy of using attenuated mutants for preventing S. pneumoniae infections. For example, the pabB deletion strain is very rapidly cleared from the mouse nasopharynx and was only weakly immunogenic after nasopharyngeal administration.¹¹² Repeated dosing of poorly colonizing strains may overcome this issue.^{82,107,110,115}

Another potential problem is the surprising lack of cross-protection induced by attenuated strains in some of these studies. For example, although the protein antigens targeted by protective responses are largely conserved between S. pneumoniae strains, adaptive responses to one episode of colonization or systemic vaccination with a live attenuated vaccine either were not or only weakly cross-protective against heterologous strains in mouse models.^112,115 The reasons for this poor cross-protective immunity are not clear and require further investigation to ensure that nasopharyngeal administration with a single strain can provide the broad heterologous protection required for an effective vaccine.

Reversion to wild type with loss of the attenuating mutation is a significant safety risk in the use of live attenuated bacteria as vaccines. This is a particular concern for pathogens such as S. pneumoniae that are naturally competent and are known to undergo recombination events during colonization. To avoid this, attenuated strains would need to contain at least two independent mutations. Another strategy to mitigate this risk would be to delete the competence machinery, rendering the strain unable to uptake foreign DNA and thereby preventing recombination events with the resident nasal flora which may possess similar virulence factors. In addition, the effect of administration of genetically modified bacteria to the nasopharynx on the existing nasal flora is not known, and will need to be evaluated carefully to ensure there are no unforeseen deleterious consequences.

Heterologous expression of protective antigens

An alternative approach has been to express recombinant protein in nonpathogenic species. Such strategies provide effective protection at the mucosal surface and during invasive disease.¹¹⁶ Lactic acid bacteria (LAB) are commonly used to manufacture foodstuffs and are therefore a safe alternative which are also known to elicit systemic and mucosal responses.¹¹⁷ The LAB, Lactobacillus casei, has been developed as an intranasal vaccine which expresses the S. pneumoniae protein antigen PspA and induces antibodies that protect mice from a systemic challenge.¹¹⁸ Another LAB, Lactococcus lactis, has also been used to express S. pneumoniae protein antigens including PspA, PpmA, PsaA, PppA, and SlrA¹¹⁹ or serotype 3¹²⁰ and serotype 14¹²¹ capsular polysaccharides. Colonization with the L. lactis strains expressing S. pneumoniae capsules led to the induction of specific IgG and IgM antibodies.^120,121 The oral commensal species Streptococcus gordonii, which also stimulates mucosal immunity,¹²² has been engineered to express protective antigens from S. pyogenes which are immunogenic in mouse models when inoculated intranasally and orally.¹²³ S. gordonii was also investigated as a means to express N. meningitidis antigens which induced bactericidal antibodies in intranasally immunized mice¹²⁴ and Bordetella pertussis antigens which when used in oral colonization induced systemic and mucosal antibodies.^122,125 These animal models and early studies in humans indicate that S. gordonii is a suitable vector for presenting heterologous antigens for a colonization approach vaccination strategy.¹²⁶ However, a limitation of this strategy is the use of a limited number of antigens, which could restrict the range and strength of any protective immune response. Nevertheless, these early results indicate that this may be an area of potential future development applicable to a number of bacteria species, perhaps in combination with the use of closely related nonpathogenic species discussed earlier. For example, the expression of important N. meningitidis antigens in N. lactamica could increase the strength of cross-protective immunity induced by colonization with the modified N. lactamica strain.

Overview and future directions

Colonization of the nasopharynx is central to disease development and adaptive immune responses to potentially pathogenic organisms. Modulation of host–pathogen interactions at this site could be a powerful method of preventing serious bacterial infections for a range of common pathogens. There are several important characteristics which an attenuated microorganism must have to serve as a potential live human vaccine: 1) mutations must be stable and severely attenuate virulence to prevent the strain from causing lung or systemic infection; 2) two or more virulence genes should be mutated to minimize the chance of revertants developing; 3) the attenuated strain should retain the ability to stimulate significant increases in adaptive immune responses after nasopharyngeal administration, including mucosal immune responses that prevent lung infection; and 4) adaptive immunity to the mutant strain should result in cross-strain protection. Currently, most data showing the utility of nasopharyngeal colonization with attenuated or nonpathogenic organisms as a vaccination strategy have been obtained using animal models. However, the development of human models of nasopharyngeal carriage^{104,127–129} now allows the strategies using colonization to prevent infection to be tested in humans and to assess whether their potential can be fulfilled.

Disclosure

The authors report no conflicts of interest in this work.

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