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REVIEW ARTICLE

New Biological Insights in the Immunomodulatory Effects of Mucosal Polybacterial Vaccines in Clinical Practice Marta Tejera-Alhambraa, Oscar Palomaresb, Rebeca Pérez de Diegoc, Ignacio Díaz-Lezcanoc,d and Silvia Sánchez-Ramónc,d a

INMUNOTEK, SL, Madrid, Spain; bChemistry Faculty, Complutense University School of Chemistry, Madrid; cDepartment of Clinical Immunology and IdISSC, Hospital Clínico San Carlos; and dDept. of Microbiology I, Complutense University School of Medicine, Madrid, Spain

ARTICLE HISTORY Received: July 1, 2016 Accepted: August 26, 2016 DOI: 10.2174/138161282266616082 9143129

Abstract: A main focus in healthcare is the active search for alternative strategies to antibiotics, both for prophylactic and therapeutical interventions, due to the accelerated and widespread increase in antibiotic resistance. This problem is more marked for patients with recurrent infections, in which the risk for antibiotic resistance and adverse effects is higher and can be life-threatening. Although antibiotics remain the mainstay of treatment for infectious diseases, prophylactic vaccines via the mucosal route in defined populations of patients with recurrent infections has gained use in recent years. Concomitantly, relevant advances in the formulation and administration of these vaccines driven by an increased knowledge of mucosal immunity have expanded their use, although still in its infancy. These drugs target both the innate and adaptive immune systems, at the actual point of entry of most pathogens. A fascinating new application of the concepts of trained immunity may open novel studies in Silvia Sánchez-Ramón their potential uses, given the paradoxically simultaneous pro-tolerogenic and boosting effector effects on diverse immune cells for different antigens. Here we delineate an updated review on the immunomodulatory mechanisms of mucosal polybacterial vaccines.

Keywords: Mucosal immune system; mucosal bacterial vaccines; immunomodulation; recurrent infections. 1. INTRODUCTION 1.1. The Threat of Antibiotic-Resistant Bacteria Antibiotic resistant bacteria are currently in the limelight, since antibiotic-resistant infections are a major public health concern warned by the World Economic Forum and the World Health Organization [1-3]. Antibiotics are the cornerstone of therapy of infectious diseases and although they are effective against most bacterial, fungal and parasitic pathogens available, only a small number of these drugs are effective against viral pathogens. Pharmaceutical research in antimicrobial resistance is far behind other health concerns such as oncology [4] with very few new antibiotics available in the last decades [5]. There are few antibiotics that make it to the market and resistance appears shortly after or is detected even sometimes before release to the market [5, 6]. Currently, many people are dying from life-threatening invasive infections due to resistant pathogens. For instance, despite the high impact in reducing mucosal and invasive disease by Streptococcus pneumoniae (pneumococcus) due to the widespread use of antibiotics and the implementation of new parenteral conjugated vaccines, it remains a prevailing cause of morbimortality in humans [7, 8]. This resistance emerges partly through the overuse of antibiotics in humans, but is also due to the use of antibiotics in animal production systems [9] and agriculture [10]. Testing the antibiotic sensitivity of bacteria requires their isolation and culture time, thus in most cases physicians use to prescribe broad-spectrum antibiotics empirically [4] Rapid molecular diagnosis and targeted therapies against pathogens would help to slow down antibiotics resistance [4]. Methicillin*Address correspondence to this author at the Department of Clinical Immunology, Hospital Clínico San Carlos, Calle Profesor Martín Lagos SN, 2040 Madrid, Spain; Tel: +34913303000; Ext: 7170; E-mail: [email protected] 1381-6128/16 $58.00+.00

resistant Staphylococcus aureus (MRSA) and multidrug-resistant Acinetobacter baumannii complex (MDRAB) are examples of drug-resistant bacterium that are becoming increasingly prevalent worldwide [11, 12]. In addition, combination of broadly prescribed antibiotics, such as cephalosporin and quinolones, is considered a risk factor for the appearance of β-lactamase-producing resistant Escherichia coli [13]. With this picture of “post-antibiotic era”, there is an urgent need for new alternatives to the management of infectious diseases. Antibiotics will remain the mainstay for fighting bacteria, but alternative drugs that may replace them or adjuvant therapies will help to prevent these infections. In natural conditions, most bacteria live not as individual cells, but as pseudomulticellular organisms displaying communally coordinated behavior through archetypal intercellular signal molecules in a process known as “quorum sensing” (QS), that enhances the bacterial capacity for adaptation through the uptake and incorporation by recombination of new genetic material from the surrounding microbial community [14]. This coordination of the bacterial attack maximizes the chances of establishing an infection and allows bacterial dissemination [15]. Approximately 65% of human infections form bacterial biofilms, leading to chronic infections [15]. As a benefit of living within these structures bacteria in biofilms are physically protected and 10 to 1000-fold more resistant to antibiotics than single living bacteria [15]. Currently there are no antibiotics effective in curing bacterial infections forming biofilms. A particular case for risk of antibiotic resistance are those patients suffering recurrent bacterial infections that need to be treated with antibiotics almost chronically. In addition to be more susceptible to life-threatening infections, these patients may be proned to dysbiosis following the harming of normal microbiota [16], which may promote subsequent fungal superinfection. Especially in the © 2016 Bentham Science Publishers

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context of recurrent infections, mucosal polybacterial vaccines may well represent a realistic alternative to broad range antibiotics used for preventive purposes. While these vaccines are already being used to prevent infections in immunocompetent patients, their use in more susceptible immunocompromised patients is starting to be considered. In this review the current knowledge on their mechanisms of action of such vaccines will be addressed. 1.2. Mucosal Immunity The mucosae cover an extensive surface in our body, approximately 200 times larger than the skin [17]. Mucosae are the main entranceway of infectious and environmental agents. Mucosal infections are a major cause of death worldwide, with children and the elderly especially susceptible and with no available effective vaccines in most diseases. The mucosa-associated lymphoid tissue (MALT) involves a complex network of highly specialized and compartmentalized components of the innate and adaptive immune systems [17, 18]. The MALT includes the nasopharynx-associated lymphoid tissue (NALT), the bronchus-associated lymphoid tissue (BALT) and the gut-associated lymphoid tissue (GALT), the urogenital tract, as well as the exocrine glands [19]. GALT and BALT have a single-layer columnar epithelia composed of lymphoid micro-compartments as the Peyer's patches (PPs) and isolated lymphoid follicles (ILFs) (type I mucosal organized lymphoid tissue), whereas the eye, mouth and vaginal associated lymphoid tissue have a protective stratified squamous epithelial layer (type II mucosal lymphoid tissue) [20]. The oral-pharyngeal mucosa possesses a stratified squamous epithelium with specialized MALT composed of buccal mucosa, tonsils, salivary glands and lymphoid follicles [19]. In healthy individuals, almost 80% of all immunocytes are located in the MALT, accumulated or in transit across the different mucosal tissues [18]. The MALT maintains a constant dialog with the commensal bacteria and a permanent surveillance against pathogens, mediated in part by the continuous presentation of antigens by antigen presenting cells (APCs), including dendritic cells

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(DCs), macrophages, and B cells. The microbiota is considered as a real organ of the immune system, essential in the correct function of the host immunity, shaping a correct symbiosis and homeostasis [21]. The antigen uptake by DCs is performed by direct and indirect pathways [22]. The indirect pathways can be Microfold (M)-cell dependent, globet cell-dependent, neonatal Fc Receptor (FcRn) dependent and apoptosis-dependent [22]. M cells are specialized cells that carry antigen and enteric bacteria through the epithelial surface for capture and processing by the surrounding DCs and macrophages to initiate the immune response [23]. In the oral mucosa there are also M-cell-like cells responsible for luminal antigen uptake [19]. Globet-cells deliver low-molecular-weight soluble antigens to underlying CD103+ DCs in the lamina propria [24], FcRn mediates the bidirectional transport of IgG-antigen immune complexes [25] and antigens associated with apoptotic epithelial cells can be taken up by DCs [26]. DCs can also directly capture antigens from the lumen by extending dendrites [22]. APCs detect pathogens through a complex, extensive repertoire of innate receptors or pattern recognition receptors (PRRs), which interact with evolutionarily conserved pathogen-associated molecular patterns (PAMPs) to elicit an immune response. There are several families of PRRs described that intervene in host defense: Toll-like receptors (TLRs) (Fig. 1 and Table 1), cell-surface C-type lectin receptors (CLRs); cytosolic retinoic acid-inducible gene 1 (RIG-I)-like receptors (RLRs) and nucleotide-binding oligomerization domain (NOD)-like receptors (NLRs), C-type lectin receptors (CLRs) and AIM-2 like receptors, as well as the cytosolic sensors for nucleic acids, DNA and RNA receptors. Damage-associated molecular patterns (DAMPs) from injured cells can also activate pattern recognition receptors (PRRs) (Table 1). Different regulated forms of cell death, such as apoptosis (programmed and caspase-dependent cell death), and necroptosis (programmed, via receptor-interacting protein kinase-1 (RIPK1) and RIPK3) or non-regulated nonprogrammed necrosis may induce distinct signatures of DAMPs released into the extracellular space [27-29].

Fig. (1). Ligation of pathogen-associated molecular patterns (PAMPs) to their cognate pattern recognition receptors (PRRs).

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Table 1. Innate repertoire of pattern recognition receptors (PRRs) binding to pathogen-associated molecular patterns (PAMPs) and to danger-associated molecular patterns (DAMPs) [27-29]. PAMPs (Ligand)

Expressed/induced by

PRRs TLR1, TLR2,

Lipoproteins,

Eubacteria

peptidoglycan,

Parasites

zymosan,

Fungi

lipoarabinomannan, porins, GPI-mucin, phospholipomannan, β-glycan tryacil lipopeptides,

Dectin1/2

diacyl lipopeptides, lipoteichoic acid

TLR2/TLR1, Mycoplasma

TLR2/TLR6

Diaminopimelic acid,

Gram-ve bacteria

NOD1

Muropeptides

Gram +ve and –ve bacteria

NOD2, NALP1 and NALP3

Envelope glycoproteins, glycoinositolphospholipids, mannan,

Viruses

TLR4

Protozoa

HSP70

Candida Host

Lypopolysaccharydes

Gram –ve bacteria

TLR4, DC-SIGN, CD14, CR3 and CR4

Parasites Fungi Unmetylated CpG ODN (ssDNA)

Most bacteria

TLR9, DHX9, DHX36

Viruses Fungi, protozoa dsDNA

Viruses

DAI, IFI16, STING, AIM2, DDX4, MRE11, cGAS

Flagellin

Motile bacteria

TLR5

dsRNA

Viruses

TLR3, RIG-1 helicase, MDA5

ssRNA

Viruses

TLR7, TLR8

23SrRNA

Bacteria

TLR13

Carbohydrates

Viruses

C-type lectins, mannose receptor

Fungi Mycobacterium Profilin

Protozoa

TLR11

DAMPS HMGB1

TLR2, TLR4, and RAGE DNA

HSP20, HSP60, HSP70, HSP90, and HSP100

infections, wounding, or heat

LR2 and TLR4

S100 proteins (S100A8, S100A9, and S100A12)

accidental necrosis

RAGE and TLR4

Galectins (1 and 3)

damaged or dead cells

α- and β-defensins

infection

TLR4

Cathelicidins

infection

TLR7, TLR9, and RAGE DNA

Calreticulin

apoptotic cell death

Mitochondrial DNA

RAGE, TLR9

NFPs Adenosine 5'-triphosphate

recognition signal for phagocytosis FPRs 1, 2 and 3.

apoptotic and necrotic cell death

NLRP3

TLR: Toll-like receptor; NOD: nucleotide-binding oligomerization domain; NALP3 – NACHT, LRR and PYD domains-containing protein 3; DC-SIGN: dendritic cell-specific intercellular adhesion molecule-3-grabbing non-integrin; CR3/4: complement receptors; DHX: DHX–DexD/H-box helicase; DAI: DNA-dependent activator of IRFs; IFI16: interferoninducible protein 16; STING: stimulator of interferon genes; AIM2: absent in melanoma; DDX4: DEAD (Asp-Glu-Ala-Asp) box polypeptide 4; MRE11: Meiotic Recombination 11 Homolog; cGAS: cyclic-GMP-AMP (cGAMP) synthase; RIG-1: retinoic acid-inducible gene 1; MDA5: melanoma differentiation-associated protein HMGB1: High mobility group box 1 protein; HSP: Heat shock proteins; NFP: N-formylated peptides; FPRs: formyl-peptide receptors.

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After antigen uptake, APCs (Langerhans cells, DCs and macrophages) in the oral mucosa, which reside in the epithelium or lamina propria, migrate into MALT and draining lymph nodes and initiate the adaptive immune response by stimulating T cell proliferation and differentiation [19]. The stimulated B and T lymphocytes leave the site of initial antigen presentation (inductive mucosa) and transit through the lymph and arrive to mucosal sites (effector mucosa) where they can differentiate into memory of effector cells [18]. This migration is largely mediated by tissuespecific integrins and chemokines [30, 31]. DCs are key initiators of the adaptive immune response and vaccine-induced specific immunity. There are diverse subsets of precommitted DCs at different stages of maturation that induce the expression of different transcription factors (T-bet, GATA3, RORgt or Foxp3) and cytokines (IFNγ, IL-4, IL-17, TGFβ, IL-35 and IL10), leading to the clonal expansion and the differentiation of these cells into T-cell subpopulations (Th1, Th2, Th17 or regulatory T cells, Treg). The CX3CR1+ DC subset induces the differentiation of T cells from the Th1 to the Th17 subtype [32], whereas CD103 + DCs induce T-cell differentiation into Treg in the lamina propria of the colon in mice a process partly driven by retinoic acid and TGFβ [33-35]. At the same time, T cells are directed to submucosal regions, where they perform their effector functions. Mucosal vaccines administered through the intranasal route can generate protection against enteric pathogens. As one mechanism involved, lungderived DCs are able to up-regulate the gut-homing integrin α4β7 and induce the migration of T cell to the gastrointestinal tract, protecting against enteric Salmonella infection [36]. DCs and Th0 cells also interact with B cells and promote their differentiation and antibody production within the mucosa. Most immunoglobulin (Ig) isotype class switching is dependent on T cells, but there are T-cell independent processes, where B cells in mucosal tissues become IgA secreting plasma cells by the direct stimulation of BAFF and/or APRIL secreted by epithelial cells or DCs [37, 38]. The magnitude of the immune protection conferred by the stimulation of mucosal and systemic antibody production by mucosal vaccines has been investigated in very few studies [16]. The role of follicular helper T cells in mucosal immunity has been recognized for some time, but the roles of Th17 cells and IL-17 during infection and in vaccineinduced immunity has only recently been elucidated. CD103+ CD11b+ DCs are the major migratory DC population and may induce Th17 differentiation when TLR stimulated [39]. In this sense, filamentous bacteria present in the commensal microbiota can stimulate Th17 differentiation [40] following the stimulation of CD103+CD11b+ DCs through the TLR5 agonist flagellin. These DCs do not only promote the differentiation of Th17 but also of Th1 lymphocytes and immunoglobulin A plasma cells through the production of retinoic acid [41]. Recently another subtype of DCs CCR2(+)CD103(-) DCs present in the human intestine has been identified to drive interleukin IL17 production by T cells in vitro [42]. Some studies have demonstrated a requirement of IL-17 for the protective effect induced by vaccines, including those against S. pneumoniae, M. tuberculosis and influenza [43-46]. Furthermore, IL-17 increases secretory IgA (sIgA) levels by increasing the expression of polymeric Ig receptor in the epithelial mucosa [47], and promotes the differentiation of T cell-dependent B cells into IgAsecreting plasma cells [48]. These data confer a relevant role for IL17 in mucosal immunity. Th17 responses can induce protective immunity against many bacterial infections including those caused by antibiotic-resistant metallo-beta-lactamases Klebsiella pneumoniae (Reviewed in [49]). Vaccine strategies using specific cell populations involved in the transport of antigens can induce the development of Th17 cells and/or the production and transport of sIgA, thereby optimizing the protective immune response at the mucosal surface. An increase in the production of sIgA and its trafficking to epithelial cells prevent pathogen invasion and promote immunity at the mucosal surface.

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1.3. Mucosal Vaccines at the Frontier of Immunity and Tolerance Unlike the more conventional parenterally administered vaccines, mucosal vaccines target directly the specific tissues where pathogens initiate or disseminate infections. At mucosal surfaces there is a fine immunological balance maintained by the commensal flora. A successful mucosal vaccine must prevent the local infection by favoring the induction of an immunological response at the damaged mucosa with the aim of restoring the homeostasis through the stimulation of both innate and adaptive responses. The induction of innate responses would involve the activation of DCs, macrophages, epithelial cells and NK cells, whereas the adaptive immune system would involve the production of specific antibodies and of memory T and B cells [16]. Appropriately formulated mucosal vaccines stimulating all arms of the immune system thus appear to be promising tools for the treatment and prevention of infections, mostly at mucosal sites [50-53]. The mucosal antibody responses elicited by these vaccines inhibit a critical step in microbial pathogenesis, the adhesion of the microbe to the mucosal epithelial cells, conferring better protection against colonization and invasion challenges than conventional parenteral vaccines [54]. Mucosal IgG can mediate the opsonization and internalization of pathogens that have breached the epithelial barrier by phagocytes. This leads to phagocyte activation and the killing of the pathogen or the presentation of pathogen-derived antigens, activating multiple subsets of mucosal effector T cells, such as Th17 cells, γδT cells and CD8+ CTL cells, to promote pathogen containment or clearance [55]. Tissue-resident memory T cells (TRM) cells reside in mucosal tissues and can respond rapidly to pathogen infection locally, independently from T cells recruitment from the blood [56]. CD8+ TRM cells when activated release IFN-γ that amplify and complement the activation of the innate immune system, creating an antiviral microenvironment in the mucosa. The induction of TRM cells is a current challenge for vaccination strategies and mucosal vaccines might possibly be an effective route to generate persistent TRM cells [56]. Mucosal vaccines must reach a fine balance between stimulation and tolerance of the mucosa. The induction of oral tolerance by the mucosal administration of vaccines remains a major challenge in the development of mucosal vaccines. Sublingual vaccines for allergen immunotherapy have been used for almost forty years and recent studies suggest that sublingual immunotherapy may enhance the induction of tolerance. According to the current model of the underlying immune mechanisms, the allergen is taken up by tonsil plasmacytoid tolerogenic dendritic cells, which then migrate to the cervical lymph nodes and induce Treg responses [57]. Biopsies of the oral epithelium have demonstrated more Treg cells in SLIT treated patients than in placebo-treated patients [58]. Autoimmune diseases are a major target for the development of therapeutic vaccines [59]. The generation of sIgA has been reported to involve multiple regulatory mechanisms that also promote mucosal and systemic T-cell hyporesponsiveness by inducing Treg cell differentiation [16]. Epithelial integrity of the mucosae depends on the interaction with microbial components from the host microbiota and environment, the secretion of IgA and Treg cells [60]. The mucosal administration of antigens may induce a profound suppression in cellular immunity, as demonstrated by the weaker delayed type hypersensitivity reactions, lower levels of T-cell proliferation and higher levels of T-cell immunosuppressive cytokine secretion in the absence of an effect on humoral immunity [57, 61, 62]. A study showed that oral vaccination with an attenuated Salmonella vaccine strain expressing colonization factor antigen I (CFA/I) fimbriae elicited fimbria-specific Treg cells and protected against experimental autoimmune encephalomyelitis (EAE), a murine model of multiple sclerosis [63]. In this study, the oral vaccination against an irrelevant antigen induced Treg cells that could control autoimmunity. Another recent study in EAE has shown that

New Biological Insights in the Immunomodulatory Effects of Mucosal Polybacterial

a bacterial infection of the respiratory tract can attenuate the disease by promoting production of the anti-inflammatory cytokine IL-10 which may suppress migration of self-reactive T cells in the lungs into the central nervous system [64]. These studies, and also others performed in experimental rheumatoid arthritis, suggest that inducing a bystander immunity against bacteria could potentially control autoimmunity or inflammatory processes [59, 61]. This immunomodulatory effect of some bacteria may be exploited to rise a new concept of bacterial mucosal vaccines as a therapeutic approach to favor immune tolerance and homeostasis in the context of autoimmune diseases. 1.4. Trained Immunity and Mucosal Bacterial Vaccines: Mechanisms for Pseudo-Specific Efficacy? Mucosal vaccines with whole inactivated bacteria or bacterial lysates stimulate the immune system resulting in lower rates of reinfections from the same pathogens or to other pathogens than those contained in the vaccines [65-67]. The underlying mechanisms of these collateral beneficial clinical effects may in part be linked to a recently described form of innate memory or trained immunity. Besides, the stimulation of the innate immune cells in the mucosa is able to polarize the adaptive immune response. Traditionally, the innate immune system response has been considered to be nonspecific, rapid and identical at every encounter with a pathogen, without memory capability [68]. However, recent cumulative evidence of memory traits in the innate immune system has changed this paradigm. The term trained immunity has been recently coined [69] to define a heightened response to a secondary infection that can be exerted towards the same microorganism or to a different one (cross-protection) (Fig. 2) [69-71]. In plants and invertebrates, which lack an adaptive immune system, this immunological mem-

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ory was primarily observed [72]. Plants have a resistance upon reexposure that spreads from the site of infection through the whole plant, named systemic acquired resistance (SAR), which protects them from different pathogens for long periods [73, 74]. Numerous studies in invertebrates demonstrate an immunological memory that protects them against secondary infections from nonspecific pathogens and they even show allograft rejection after tissue transplantation (for review, see [72] and [69]). In mammals, studies from fifty years ago already described how infection conferred acquired antibacterial properties toward specific and unrelated bacterial pathogens [75]. More recent studies have demonstrated that innate immune cells such as monocytes, macrophages and NK cells display memory characteristics upon reinfections that provide protection against certain infections independently of T and B lymphocytes [76-79]. The mechanisms that mediate this trained immunity include epigenetic and transcriptional reprogramming, including histone and DNA modifications that favor the transcription of proinflammatory cytokines [76], contribution of microRNAs, as well as metabolic changes [80]. Finally, all these mechanisms may improve pathogen recognition by the up-modulation of PRRs on the surface of innate immune cells and better host adaptation [69]. The reduced overall children mortality in those vaccinated with measles and tuberculosis vaccines, has been observed in randomized trials (reviewed in [81]). This positive outcome cannot be exclusive to specific protection to these diseases and there are immunological studies suggesting that these non-specific effects are mediated through trained innate immunity and its interaction with the adaptive immune system [82]. Jensen et al. [82] have recently demonstrated in a randomized-controlled trial with low-birth-weight infants early vaccinated against tuberculosis with Bacillus Calmette-Guérin (BCG) that four weeks after immunization, BCG-vaccinated infants had a significant increased production of pro-inflammatory cytoki-

Fig. (2). Classical view of innate immunity as a rapid, unspecific and effective response lacking memory. A new paradigm challenging this view states that innate cells, such as monocytes, dendritic cells or NK cells are able to change after a first encounter with a pathogen A through epigenetic reprogramming and metabolic glycolisis induction, which will induce a more effective response to a different pathogen B. This pattern of innate memory has been called trained immunity and goes parallel to induction of the adaptive specific response by the same cells. Figure adapted from [124].

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nes IL-1β, IL-6, TNF-α and IFN-γ after stimulation with heterogeneous challenges for Toll-like receptors. This early immunization of the neonatal immune system with Th1 polarizing cytokines and monocyte-derived pro-inflammatory cytokines may accelerate the development of the immune system and mediate in the protection against infections [82] by increasing the functional efficiency of the immune system upon a re-challenge. Interestingly, trained innate immunity has also been observed during the course of a natural viral infection. A recent study in newborn infants of HBV-infected mothers has shown that HBV exposure in utero triggers a state of trained immunity with an enhanced innate immune cell maturation and increased Th1 development. These features of a more developed immune system resulted in a better ability to respond to unrelated pathogen exposure with increased production of proinflammatory cytokines such as IL-12P40, IFN-γ, and TNF-α after stimulation with unrelated bacterial challenges [83]. Zhang et al. [84] have demonstrated how the treatment with bacterial flagellin is able to prevent and cure the chronic infection with rotavirus with mechanisms related to innate immunity. The protector effect of bacterial derived immunomodulators in an influenza murine model has also been described [85]. Mucosal polybacterial vaccines boost both the innate and the adaptive immune system. These vaccines are administrated for their immunostimulant capacity for the management of recurrent respiratory tract infections, prevention of exacerbations in chronic obstructive pulmonary disease (COPD), recurrent wheezing and acute respiratory infections, and recurrent urinary tract infections [65-67, 86, 87]. The effects of these bacterial mucosal vaccines containing bacterial lysates (OM-85) are attributed to a boost of the innate immunity (increased the phagocytic and metabolic activity of macrophages and upregulated adhesion molecules) as well as an enhanced Th1 cellular response accompanied of an increased humoral response (mostly increased secretory IgA levels) (Reviewed in [86, 88]). In human DCs, this bacterial preparation selectively induces NF-ĸB and MAPK with strong upregulations of chemokines (CXCL8, CXCL6, CCL3, CCL20 and CCL22) and cytokines (IL-6, BAFF and IL-10). In vivo experiments performed in mice have proven the cross-protection conferred by orally administered OM85 to unrelated pathogens such as Salmonella typhimurium and influenza virus infection. This non-specific protection can be an example of trained immunity with heterologous immunological effects boosting the innate and adaptive immune system to a more efficacious response [89]. A pilot clinical study evaluated the clinical and immunological effects of mucosal whole-bacterium sublingual vaccines on antigenspecific immune responses to bacteria responsible for respiratory tract infections [67]. Daily immunization with a sublingual polyvalent bacterial preparation (Bactek®) over a period of six months was studied in a series of 17 patients with RRTI. The rate of respiratory infections in immunized patients significantly decreased compared to that in the previous year. It was also observed that the severity and duration of the infectious respiratory events was diminished. Immunological results showed a significant increase in the frequency and proliferative capacity of specific CD3+CD4+ T cells against bacterial antigens contained in the vaccine, was observed at 6-months of treatment together with a significant increase in the frequency of CD3+ T cells. In addition, an increase in the proliferative capacity of CD3+CD4+ and CD3+CD8+ T cells specific for influenza antigens was also observed after 6-months of treatment, which might suggest that the bacterial preparation had stimulated the immune system in a non-specific manner to T lymphocytes primed in vivo with the influenza virus they gave a more potent and robust response. Very interesting is the particular case of one patient in this study suffering from 12 episodes of oral herpes the previous year that reported 3 episodes after immunization with the bacterial preparation, suggesting an in vivo non-specific or by-

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stander effect boosting antiviral responses. In another study with 669 women with recurrent urinary tract infections (RUTIs) that compared the efficacy between the prophylactic treatment with antibiotics or with the sublingual bacterial preparation (Uromune®), all patients in the antibiotic group experienced a new infection in the next year versus less than 10% in the group treated with the bacterial preparation. An interesting additional finding in this study was that patients that previously had infections with bacteria not included in the vaccine did not develop re-infections, suggesting a cross-protection that could be attributed to a heightened innate immune response. These non-specific immunomodulatory properties of bacterial mucosal vaccines is of crucial clinical interest, preventing the recurrence of infections against specific pathogens contained in the vaccine, but also to protect individuals against unrelated infections or pathogenic insults. Thus, such vaccines could increase host defenses against microorganisms other than the bacteria included in the vaccine, including viruses, and fungi [90-92]. 1.5. Polybacterial Mucosal Vaccines: Advantages of the New Formulations Several different types of mucosal bacterial vaccines have been developed for the prevention of recurrent bacterial infections, both at the initial stages of disease (colonization and infection with pathogens) and by blocking disease development [93]. These vaccines may contain soluble antigens (lysates or bacterial components) or inactivated whole bacterial cells from one or more species or strains, in different formulations. Polybacterial mucosal vaccines have different advantages over conventional parenteral vaccines (Fig. 3): i) They exert their immunological effects directly at the site of infection in the mucosa and they can prevent the infection and pathogen colonization; ii) immunization at one mucosal can result in antibody secretion in other distant mucosa as well as systemically [18]; iii) they do not require medical personnel for their administration and can be used in mass vaccination programs and disease prevention campaigns; iv) they are painless and easy to administer. However, unlike parenteral vaccines, they require a repetitive stimulus and the duration of the treatment should be longer. They require further development both scientifically and clinically. Depending on the route of administration, the effect of the vaccine may be different. For instance, with oral vaccines, the bioavailability is reduced as antigen may be partly degraded by digestive enzymes. The dose of mucosal vaccine that enters the antibody is difficult to be measured accurately [16]. Mucosal vaccines with whole inactivated bacteria have the advantage of having naturally articulated antigens that will facilitate their efficacious recognition of APCs through PRR and the efficient uptake by M-cell-like cells. Inactivated whole bacteria stimulate more consistently the immune system than soluble bacterial lysates [94], as several PAMPs can be detected in the microbial cell and the phagocytosis with full microorganism ensures more complete responses [95]. Phagocytosis of whole bacteria enables their acid digestion in the lysosomes and the liberation of bacterial ligands that trigger intracellular mediated TLR responses. This phagocytosis is essential for initiating MyD88 dependent responses and the secretion of cytokines [95]. Interestingly, Gram+ bacteria stimulate the production of IL-12, whereas Gram (–) bacteria stimulate the production of IL-10 by monocytes. The combination of Gram (+) and Gram (–) bacteria in polybacterial mucosal vaccines seems to provide a synergistic immunological responses that should be further explored depending on the pathology involved [96]. Formulations based on combinations of inactivated whole bacteria boost the host immune response more effectively [97, 98]. Polybacterial mucosal vaccines may have a higher likelihood of success than a single-bacterium approach in boosting the immune system in order to promote the elimination of infections. Indeed,

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Fig. (3). Balance of advantages and disadvantages of mucosal vaccines formulations. (McA: Mechanism of action)

polyvalent bacterial preparations have been shown to stimulate phagocytosis, sIgA synthesis, and the production of surfactant, IFNγ and Th1 type cytokines, and such preparations have proved successful for the treatment of recurrent respiratory, urogenital and periodontal infections. Recurrent infections may be associated with specific functional defects of immunity to inhaled pathogenic bacteria, such as poor bacterial clearance, leading to chronic overt or subclinical infections [90, 99]. In this setting, polybacterial mucosal vaccines may prove useful as antimicrobial treatments, given that they are able to stimulate both innate (PRR stimulation, recruitment of NK cells, DC maturation) and adaptive responses. In summary, mucosal immunization with fully inactivated whole bacteria directly stimulates the immune components present in the mucosa [100] and provides greater clinical benefit, for several reasons: i) whole bacteria are presented to the immune system in a more natural way, maximizing their full potential as immunogens [92, 101, 102] ii) whole bacteria stimulate different immune mechanisms that have been shown to be very important for a full cell activation, such as phagocytosis [95]. 1.6. Improved Delivery Routes for Polybacterial Mucosal Vaccines Classic mucosal polybacterial vaccines are administered orally, to target the intestinal mucosa. Following vaccination via this route, the main mucosal immune response is local and regional, with limited access to distant sites [18]. In the last years, new delivery routes have been developed to enhance the mucosal immune response at distant sites at which infection occurs. Nasal immunization triggers not only local and regional mucosal immune responses, but also an immune response at distant sites, inducing a strong urogenital mucosal immune response. This route has proved effective for stimulating the respiratory immune system at the actual point of entry of most pathogens, inducing both humoral and cellular responses to antigens [103, 104]. As via nasal immunization, antigens are not exposed to degradation by digestive

enzymes, this route requires much lower doses of antigen than oral vaccination for the induction of antigen-specific mucosal immune responses. However, antigen administration via the nasal mucosa does not fully stimulate the NALT (nasal associated lymphoid tissue). Efforts have therefore focused on the development of an antigenic intranasal vaccine that stimulates the NALT. However, this pathway has been associated with adverse nervous system effects, such as Bell’s palsy [105, 106]. The sublingual route has a potential utility that has been demonstrated in a number of studies [107, 108]. This route is able to stimulate the distant mucosa (respiratory, genitourinary and gastrointestinal tract[107]) and also confers a systemic immunostimulation. Nagai et al.[109] have identified that in mice sublingual antigens can be transported from ductal epithelial cells to ductal to DCs ductal. This route is safe and highly effective and induces a robust immune response similar to that induced by nasal stimulation and superior to that achieved with the oral pathway [108, 110]. Sublingual immunization induces protective immunity mediated by systemic and mucosal humoral and cellular responses [18]. The strength of sIgA induction by the sublingual route is similar to that achieved via the nasal and oral routes; vaccination via this route also induced the production of specific sIgA in the intestine, which was not observed following nasal administration [107]. Sublingual immunization has several advantages over other routes of immunization: no medical personnel is required for its administration, sublingual vaccines are easier to administer than nasal vaccines, this route has an advantage over the oral route that the antigen is not subject to significant proteolytic degradation by digestive enzymes [51, 107]. The efficacy and persistence of the immune response induced by sublingual immunization with various adjuvants suggest that this pathway is a promising alternative to immunization via other mucosal routes [50, 65, 111]. In clinical practice, administration of polybacterial vaccines sublingually has shown high clinical efficacy in the context of recurrent respiratory and urinary tract diseases [6567].

8 Current Pharmaceutical Design, 2016, Vol. 22, No. 00

Sublingual immunization is similar to nasal immunization in terms of magnitude, breadth and anatomic dissemination of the immune responses induced. Currently, a new generation of polybacterial mucosal vaccines has been developed for sublingual administration [65, 67]. Importantly, by contrast to nasal administration, sublingual administration did not lead to the redirection of antigens and/or adjuvants to the brain. INDICATIONS FOR POLYBACTERIAL VACCINES Polybacterial vaccines are currently prescribed for the prevention of infectious diseases at different mucosae, like respiratory tract [67, 112-115] or in the urinary tract [65, 66, 116]. These include the prevention of wheezing attacks in children [117] and exacerbations in adults with COPD [118]. The clinical efficacy of bacterial lysates in the prevention of respiratory tract infections in children is estimated in around 37% [119]. A similar efficacy has been found for bacterial products in the prevention of wheezing attacks in children [117]. In the prophylaxis of RUTis the clinical efficacy of bacterial lysates in adults is of 36% [120]. The efficacy of polybacterial sublingual vaccines with whole inactivated bacteria in the prevention of RUTIs has been higher than that of prophylaxis with antibiotics [65, 66]. The spectrum of patients with recurrent infections is very broad and there are subsets of patients that are especially at high risk, as those with primary and secondary immunodeficiencies. The use of fully inactivated polybacterial vaccines in primary immunodeficient patients is started to be used in the clinics [121]. The rationale behind this is that these preparations induce a broad immunostimulation in these patients that can counteract their immunodeficiency. In this sense, Alsina et al.[122] have recently described in patients with deficit in the MyD88 pathway how the stimulation of peripheral blood mononuclear cells with whole inactivated bacteria activate NF-kB, a result not seen with individual TLR agonists. This may indicate a broader inmunostimulation spectra for whole bacteria than for bacterial lysates. Immunodeficiencies secondary to immunosuppressive therapy (organ transplantation, rheumatological disorders, oncology), chronic metabolic diseases (diabetes) or infections (HIV) are conditions also prone to recurrent infections. The use of polybacterial vaccines in patients that are actively immunosuppressed to preserve a transplanted organ, or to hold under control an autoimmune disease represents a clinical challenge [123]. The immunostimulation promoted with the polybacterial preparation might by deleterious in these cases, by overcoming the immunosuppression required for their condition. However, polybacterial sublingual vaccines are currently being introduced in the management of patients with systemic autoimmune diseases and renal transplantation suffering recurrent bacterial infections without negative safety results (unpublished data). 3. CONCLUSION AND FUTURE PROSPECTS Mucosal vaccines capable of eliciting mucosal and systemic immune responses are a potentially useful tool for preventing recurrent mucosal infections, the main entry for most pathogens. New prophylactic and therapeutic strategies based on the use of mucosal vaccines consisting of whole bacteria, bacterial lysates or their derivatives, represent a significant breakthrough. By stimulating the innate immune response and generating specific mucosal and systemic memory, they provide resistance against different pathogens. Optimal efficacy may depend on their formulation and administration route. CONFLICT OF INTEREST MTA is employed by Inmunotek. The authors declare that the review was conducted in the absence of any potential conflict of interest.

Tejera-Alhambra et al.

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