Review: Key virulence factors of S. pneumoniae and non-typeable H. influenzae

Key virulence factors of Streptococcus pneumoniae and non-typeable Haemophilus influenzae: roles in host defence and immunisation

R Anderson, C Feldman

Ronald Anderson, Medical Research Council Unit for Inflammation and Immunity, Department of Immunology, Faculty of Health Sciences, University of Pretoria and Tshwane Academic Division of the National Health Laboratory Service, Pretoria, South Africa. Charles Feldman, Division of Pulmonology, Department of Internal Medicine, Charlotte Maxeke Academic Hospital, Faculty of Health Sciences, University of the Witwatersrand, Johannesburg, South Africa. Correspondence to: Dr R Anderson, Department of Immunology, P O Box 2034, Pretoria 0001 South Africa. E-mail: [email protected]

Identification and prioritisation of candidate antigens on which novel vaccines can be based, or the efficacy of existing vaccines improved, are critically dependent on characterising the strategies utilised by microbial pathogens to evade host defences, and, in particular, the key virulence factors involved. In this review, we have focused on the immune evasion strategies utilised by two important bacterial respiratory pathogens, viz Streptococcus pneumoniae and non-typeable Haemophilus influenzae, with particular emphasis on key virulence factors and their potential to serve as candidate immunogens.

Peer reviewed. (Submitted: 2010-05-17, Accepted: 2010-08-27). © SAJEI South Afr J Epidemiol Infect 2011;26(1):6-12

Introduction resistance. Importantly, both the pneumococcus and NTHi, by the mechanisms described below, adhere to and invade airway epithelium. This review will focus on two bacterial pathogens, viz Streptococus Biofilm-encased aggregates, in which both pathogens may coexist, pneumoniae, the most common aetiological agent in community- either attached to the epithelial surface, or sequestered intracellularly, acquired pneumonia, and non-typeable Haemphilus influenzae (NTHi), providing a potential mechanism for bacterial persistence.4 Concealed which, although less common, has taken on greater clinical significance in biofilm, in which they communicate by quorum sensing mechanisms, in the post-H. influenzae serotype b (Hib) conjugate vaccine era. these microbial pathogens can re-emerge at times when host defences Indeed, most invasive H. influenzae diseases in developed countries are transiently or irreversibly compromised, as may occur during infection are now caused by NTHi strains.1 This represents a potential problem with influenza virus/respiratory syncitial virus or HIV-1, respectively. for developing countries because of the inclusion of Hib in childhood vaccination programmes. Both of these bacterial pathogens possess a Key non-protein virulence factors of S. pneumoniae range of protein and non-protein virulence factors which are essential for both nasopharyngeal colonisation and progression to invasive disease. The antiphagocytic polysaccharide capsule is generally recognised as Notwithstanding current vaccines based on pneumococcal capsular being the major virulence factor of the pneumococcus, promoting resis- polysaccharides, many of these virulence factors are also candidate tance to opsono-phagocytosis and entrapment by mucopolysaccharides antigens for vaccine development. Interestingly, a pneumococcal present in mucus in the non-immune individual.5 Although weakly capsular polysaccharide conjugate vaccine utilising an NTHi carrier immunogenic, capsular polysaccharides are nevertheless the primary protein is a possible future strategy to provide dual immunoprophylaxis determinants of immune-mediated, type-specific protection against the 2 against these microbial pathogens. pneumococcus. They initiate the production of secretory immunoglobu- In the case of both of these bacterial pathogens, colonisation is mostly lin (Ig)A, an antibody which antagonises adhesion of the pneumococcus asymptomatic, but is nevertheless the first step in the pathogenesis of to respiratory epithelium, as well as production of both mucosal and invasive disease, which may be precipitated by infection with respiratory circulating IgG antibodies which promote opsono-phagocytosis. The viruses, resulting in increased expression of receptors for bacterial pneumococcus also produces copious amounts of hydrogen peroxide 3 adhesins on respiratory epithelium. (H2O2) through the action of the enzyme pyruvate oxidase, reaching low

millimolar concentrations in bacteriological culture medium. H2O2 is an Biofilm indiscrimate, cell-permeable oxidant, which is toxic for both eukaryotic

Prior to reviewing pathogen-selective virulence factors, a brief and prokaryotic cells. Importantly, H2O2 is cytotoxic for ciliated respira- consideration of the role of biofilm in microbial colonisation/persistence tory epithelium, causing ciliary slowing and epithelial damage, favour- is important, as this is a common strategy utilised by the pneumococcus ing adhesion to, and invasion of, epithelial cells by the pneumococcus.6 and NTHi to evade host defences.4 Biofilm is a hydrated, self-generated Although potentially suicidal for the pneumococcus, which does not polymer matrix in which microbial pathogens are effectively insulated produce catalase, H2O2 also appears to induce biofilm formation, which against the cellular and humoral defence mechanisms of the host. is likely to favour persistence of the microbial pathogen in the respira- Biofilm also restricts the penetration of antibiotics, favouring antibiotic tory tract.4

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Table 1: Key non-protein and protein virulence factors of the pneumococcus the cell-cell junction protein of respiratory epithelium, is a receptor for PsaA.7,12 Some serotypes possess pilus-like structures that promote Factor Activity epithelial adhesion via interaction with uncharacterised receptors, while Polysaccharide capsule Prevents binding to mucopolysaccharides, antiphagocytic. a novel pneumococcal protein adhesin has been described recently. This is the 120 kDa plasminogen- and fibronectin-binding protein B (PfbB), Hydrogen peroxide Cytotoxic, impairs mucociliary escalator function. which significantly increases the ability of the pneumococcus to adhere Phosphorylcholine Epithelial adhesin. to epithelial cells.13 Unmasking of these various pneumococcal adhesins Pneumolysin Cytotoxic, impairs mucociliary escalator function, necessitates a reduction in capsule size, with the accompanying risk of apoptosis of dendritic cells, complement consumption, increased vulnerability to phagocytosis. This risk is apparently minimised proinflammatory. by production of biofilm, a process in which bacterial neuraminidase,14 Zinc metalloproteinase Cleaves secretory IgA. and possibly H2O2, participate. These aforementioned virulence Hyaluronidase Potentiates the inhibitory effects of hydrogen peroxide mechanisms, which are summarised in Table 1, facilitate translocation and pneumolysin on mucociliary escalator function. of the pneumococcus to the lower airways, while transcytosis of the PsaA Epithelial adhesin. pathogen across the epithelial barrier via the polymeric Ig receptor

PspA, PspC Epithelial adhesin, interference with complement enables access to the bloodstream and invasion of the central nervous activation. system. PfbB Epithelial adhesin. Pneumolysin

Pneumolysin, a key protein virulence factor released upon autolysis of the Antagonism of the mucociliary escalator by H2O2, acting in concert pneumocccus, merits special mention as this toxin is critically involved in with the protein virulence factors pneumolysin and hyaluronidase as the pathogenesis of severe pneumococcal pneumonia.5,8,9,15 In addition described below,6,7 facilitates the attachment of the pneumococcus to to the adverse effects on innate and adaptive host defences mentioned airway epithelium via the binding of a third non-protein virulence factor, above, the toxin, via its cytotoxic effects on pulmonary endothelial and phosphorylcholine on the pneumococcus, to the platelet-activating alveolar epithelial cells, facilitates transmigration of the pneumococcus factor (PAF) receptor on the epithelium.8,9 from the alveoli to the interstitium, and then into the bloodstream. This, in Key protein virulence factors of S. pneumoniae turn, may lead to the development of acute respiratory failure, a serious complication of pneumococcal pneumonia.8,9,15 The pneumococcus also possesses an array of protein virulence factors which contribute to evasion and/or suppression of host defences, Using an experimental animal model, Witzenrath and colleagues provided some of which are recognised as potential vaccine candidates.8 These revealing insights into the role of pneumolysin in the pathogenesis of include the choline-binding proteins (Cbps), pneumococcal surface acute lung injury. Delivery of pneumolysin into the airways of mice proteins (Psps), neuraminidase, hyaluronidase, a zinc metalloproteinase, was accompanied by rapid, damaging effects on the alveolar capillary membrane and extracapillary vessels, resulting in vascular leakage divalent metal-binding proteins, the cholesterol-binding, pore-forming and pulmonary oedema, while infusion of the toxin into the pulmonary toxin pneumolysin, as well as the lipoprotein pneumococcal surface circulation resulted in pulmonary hypertension.16 All of these are adhesin A (PsaA). During colonisation of the nasopharynx, virulence important features of acute respiratory distress syndrome (ARDS). factors that neutralise both innate and adaptive host defences enable As mentioned earlier, these direct cytotoxic effects of pneumolysin on bacterial adhesins to promote attachment of the pneumococcus to airway cellular and structural integrity are likely to be potentiated by airway epithelium. In the case of adaptive host defences, cleavage of H O and hyaluronidase, as well as by toxin-mediated, inappropriate secretory IgA by the zinc metalloproteinase favours adhesion of the 2 2 activation/exacerbation of inflammatory responses as described below. pneumococcus, while both innate and adaptive host defences are compromised by PspA and PspC (also known as CbpA) which interfere Antipneumococcal host defences with activation of the alternative pathway of complement activation, Although nasopharyngeal colonisation by the pneumococcus is the first as well as by pneumolysin which causes depletion of complement via stage in invasive disease, it may also protect against severe infection activation of the classical pathway.8-10 As alluded to above, pneumolysin, by activating host defences.17 As mentioned earlier, IgG and secretory acting in concert with H O and hyaluronidase, also interferes with the 2 2 IgA antibodies directed against the antiphagocytic, polysaccharide protective functions of the mucociliary escalator while, in addition, this capsule of the pneumococcus are generally considered to be the primary protein toxin enables the pneumococcus to evade human dendritic cell determinants of immune-mediated type-specific protection against surveillance by interfering with the maturation of these cells, and by the pneumococcus. Antibodies to pneumococcal proteins, particularly inducing apoptosis.11 CbpA, PsaA, and pneumolysin, also confer protection which, although Notwithstanding the pro-adhesive interactions between pneumococcal less efficient, is not serotype restricted.18 Recently, several additional phosphorylcholine and the PAF receptor on airway epithelium, PspA and antipneumococcal host defence mechanisms have been identified PspC also promote attachment via binding to the epithelial polymeric which are not dependent on antibody production. These include innate Ig receptor that normally transports secretory IgA, while E-cadherin, host defences triggered by pneumolysin and lipoteichoic acid (LTA), an

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integral cell wall component, as well as adaptive responses mediated The early sub-acute, inflammatory response which contributes to by CD4+ helper T cells of the Th1 and Th17 subsets in response to the initial control of pneumococcal colonisation of the upper airways pneumococcal protein antigens, with most of the evidence derived from therefore appears to require the pore-forming actions of pneumolysin, murine models of experimental infection. as well as the interactions of the toxin and LTA/peptidoglycan with TLR-4 and TLR-2, respectively, on cells of the innate immune system, Innate antipneumococcal host defences resulting in the recruitment and activation of neutrophils and monocytes/ Depending on the local density of pneumococci in the airways, macrophages. pneumolysin, by virtue of its cholesterol-binding pore-forming activity, In addition, nucleotide oligomerisation domain (Nod)-like receptors may either protect against or promote infection. Exposure to small also contribute to innate, antipneumococcal host defences. These numbers of pneumococci leads to the production of low, sublytic are a specialised group of intracellular pattern recognition molecules, concentrations of pneumolysin which bind to airway epithelium, which, like TLRs, recognise highly conserved structures on bacterial resulting in sublethal pore formation and influx of extracellular calcium pathogens, with microbial sensing resulting in activation of cytosolic (Ca2+), membrane disruption, and osmotic stress.19-21 These events, in transcription factors and an inflammatory response.28 In the case of the turn, lead to the initiation of several intracellular signalling cascades pneumococcus, bacterial proteoglycans are recognised by epithelial Nod involving p38 and JNK mitogen-activated protein kinases, transforming proteins, specifically Nod 2.29 growth factor-b-activated kinase 1-mitogen-activated protein kinase kinase 3/6-p38 a/b, and Ca2+-calcineurin. The consequence is Antibody-independent, adaptive, antipneumococcal host activation of the ubiquitous transcription factors nuclear factor-kB and defences nuclear factor of activated T cells, with resultant initiation of synthesis Based on experimental animal and clinico-epidemiological studies, it is of various proinflammatory chemokines/cytokines, especially the now evident that antibody-independent, CD4+ T cell-mediated adaptive neutrophil chemoattractant interleukin-8 (IL-8), as well as activation of immune reponses to pneumococcal proteins also contribute to the cyclooxygenase 2, by airway epithelium. IL-8, in turn, promotes an influx natural development of immunity to pneumococcal infection, a finding 19 of neutrophils which contributes to the early control of colonisation. which has significant implications for future vaccine development.8,30,31 This early influx of these cells is also necessary for the degradation These T cell-mediated immune responses to pneumoccal protein of bacteria required for efficient delivery of pneumococcal antigen to antigens appear to be mediated by CD4+ T cells of both the Th1,32,33 22 nasal-associated lymphoid tissue and induction of mucosal immunity. and Th1730 subsets, with involvement of the cytokines IFN-g and Pneumolysin has also been reported to interact with Toll-like receptor IL-17A, respectively. Although not entirely clear, the mechanism of 4 (TLR-4), an event which contributes to both innate and adaptive protection involving Th1 cells may involve enhancement of mannose immunity to the pneumoccocus.23,24 In humans, there are currently 10 receptor-mediated phagocytosis and intracellular eradication of the recognised members of the TLR family, these being the prototype pattern pneumococcus following activation of airway macrophages by IFN-g,34 recognition molecules which function as sentinels of the innate immune as well as neutrophil influx.35 Importantly, pneumococcal capsular system. TLRs also link innate and adaptive immunity by promoting polysaccharides have been shown to bind to the mannose receptor, the up-regulation of co-stimulatory molecules on antigen-presenting as well as to other macrophage C-type lectins such as SIGN-R1.36,37 In cells, as well as inducing the production of macrophage-activating the case of Th17 cells, production of IL-17A leads to the recruitment cytokines by T cells independently of the T cell receptor (TCR) for of neutrophils and increased pneumococcal killing by these cells.30 antigen.25 Interaction of pneumolysin with airway epithelium, as well as Following initial partial control of colonisation by innate neutrophil- with other cells of the innate immune system in the airways, triggers a mediated mechanisms, recent evidence favours a primary role for Th17 signalling cascade which leads to the production of IL-1b, IL-6, IL-8, and cells in the recruitment of monocytes/macrophages to mucosal surfaces, tumour necrosis factor (TNF), all of which cooperate to promote trans- resulting in clearance of primary bacterial colonisation.30,38 endothelial migration and chemotaxis of neutrophils. This pneumolysin/ Importantly, TLRs are also expressed on naïve CD4+ T-lymphocytes, TLR-4-mediated inflammatory response may also contribute to the early as well as on central memory CD4+ T cells and Th1 effector cells, with control of pneumococcal colonisation and promote mucosal immunity. TLR-1,-5,-7, and -9 messenger RNAs having been detected in these In addition to the interactions of pneumolysin with TLR-4, LTA and cells.25,39-41 Interaction of LTA and pneumolysin with TLR-2 and TLR-4, peptidoglycan, both of which are pneumococcal cell wall components, respectively, on T cells is therefore likely to potentiate TCR-mediated are recognised by TLR-2 on cells of the innate immune system, also immune responses to pneumococcal immunogens. However, exposure resulting in the synthesis of proinflammatory cytokines.24,26 Interestingly, of T cells to TLR-2 ligands, such as LTA, also causes T cell activation engagement of TLR-4 by pneumolysin has been reported to amplify independently of the TCR, resulting in the production of IL-8 and IFN-g by TLR-2-mediated inflammation by mechanisms which remain to be naïve T cells and Th1 cells, respectively.41,42 Such a mechanism of TCR- fully characterised,24 but which may involve increased expression and independent activation of CD4+ T cells via TLR-2 (and possibly TLR-4) is sensitivity of TLR-2 on immune and inflammatory cells by an interferon-g also likely to contribute to the control of both pneumococcal colonisation (IFN-g)-driven mechanism.25 and acute infection.

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In summary, pneumococcal colonisation of the airways has several (LOS) because it lacks O-specific polysaccharide chains.47 The LPS of H. possible outcomes. Firstly, the bacteria may be eradicated by influenzaeconsists of a conserved, glucose-substituted, triheptosyl inner cooperative interactions between innate and adaptive host defence core moiety linked to lipid A, which provides the template for attachment mechanisms. Secondly, and somewhat speculatively, host defences may of variable length oligosaccharide and non-carbohydrate (phosphate initially subdue the pneumococcus, which as a survival strategy, protects substituents, O-acetyl groups) ester-linked moieties. Importantly, itself by producing biofilm, entering a quiescent phase of asymptomatic LPS shows considerable intra-chain and inter-chain heterogeneity carriage. Thirdly, in highly susceptible individuals, such as the very of glycoform structure due to the extensive LPS gene repertoire and young, the elderly, or other immunocompromised groups, colonisation variable expression profiles, with LPS phase variation being critical for may progress rapidly to acute infection. evasion of host defences and persistence.47

Adverse effects of inappropriate activation of With respect to colonisation, a majority of NTHi strains express antipneumococcal host defences phosphocholine on the LPS outer core through the activity of the Although the various host defences described above are intended enzyme, diphosphonucleoside choline transferase.47 The addition of to protect the host against the pneumococcus, hyperactivation of phosphocholine apparently enables LPS to function as an adhesin these same mechanisms may occur during acute, poorly-controlled as described below.46,47 Sialylation of LPS by sialyltransferares is an infection. The consequence is excessive production of proinflammatory additional strategy utilised by NTHi to evade host defences. Most bacterial cytokines and over-exuberant inflammatory responses. In turn, these pathogens lack sialic acid, an acidic sugar commonly expressed on cell lead to inflammation-mediated disruption of the epithelial barrier surface glycoproteins and glycolipids of eukaryotic cells. Incorporation of due to the cytotoxic actions of phagocyte-derived reactive oxidant sialic acid into LPS depends on both an exogenous source of the sugar species and proteases, favouring extra-pulmonary dissemination of and a mechanism for efficient uptake. Utilisation of sialic acid and its the pneumococcus. These proinflammatory events are exacerbated incorporation into the outer core of LPS confers several potential host by pneumolysin, which not only damages airway epithelium and evasion strategies on NTHi, viz: i) concealment of LPS epitopes that endothelium directly, but also potentiates inflammatory responses by are targets for the host immune system; ii) favours attachment of the 8,43-45 causing complement activation and hyperactivation of phagocytes. pathogen to host receptors for sialylated structures; and iii) interference Activation of antipneumococcal host defences may also pose a potential with the alternative and classical pathways of complement activation.46 hazard to HIV-infected individuals, for whom the pneumococcus is a Interestingly, phosphocholine and sialylated glycoforms of LPS appear major threat. This is because activation via TLR-2 may render naïve and to be important in biofilm formation, which, in turn, contributes to memory CD4+ T cells more susceptible to productive infection by HIV-1 persistence and disease.4,47 through increased expression of the chemokine receptor, CCR5.42 Protein virulence factors of NTHi Non-typeable H. influenzae (NTHi) Following entry of NTHi into the upper respiratory tract, the bacteria In contrast to the pneumococcus, NTHi is an unencapsulated, non- must overcome the expulsive actions of the mucociliary escalator. This toxin-producing bacterial pathogen which is less invasive than either appears to be achieved by the outer membrane proteins (OMPs), P2 the encapsulated H. influenzae serotype b (Hib) or the pneumococcus. and P5, and possibly other proteins, which promote bacterial binding to Although most commonly associated with asymptomatic carriage, NTHi mucus, while protein D, a highly conserved 42 kDa surface lipoprotein is a frequent cause of otitis media, chronic bronchitis and community- with glycerophosphodiester phosphodiesterase activity found in all 1,46 acquired pneumonia. Despite the absence of a capsule and a major strains of H. influenzae, including NTHi, possibly acting in concert with extracellular toxin, NTHi is nonetheless an astute bacterial pathogen, LPS, has been shown to impair mucociliary function.2,48 Subsequently, largely because of its propensity for genetic heterogeneity and antigenic several adhesins, in addition to phosphocholine-expressing LPS variability, enabling it to evade host defences, while complicating vaccine mentioned above, promote direct adherence to respiratory epithelium. development. In the post-Hib vaccination era, there is also increasing In approximately 80% of NTHi isolates, the major adhesins are the concern in relation to the rising prevalence of invasive disease due to related proteins, HMW1 and HMW2.49 These are high molecular weight, 1 both NTHi and non-Hib encapsulated strains. non-pilus adhesins with broad adhesive capacity, which enable NTHi to Non-protein and protein virulence factors of NTHi adhere to a variety of epithelial cell types. HMW1 appears to interact with a poorly characterised sialylated glycoprotein on epithelial cells, Like the pneumococcus, NTHi possesses a range of virulence factors while the receptor for HMW2 is unknown.48 Other protein adhesins which which promote colonisation of the nasopharynx and evasion of host promote colonisation by NTHi include: i) pili in some NTHi which bind to defences. In addition to biofilm and an IgA1 protease, these include the outer membrane lipopolysaccharide (LPS) and an array of protein components of the extracellular matrix; ii) OMPs of the autotransporter adhesins and inhibitors of innate host defences. family such as Hap, which also binds to extracellular matrix components, and Hia which interacts with an unidentified epithelial receptor; iii) the Lipopolysaccharide 16 kDa vitronectin-binding protein, protein E (PE); and iv) P5 which Lipopolysaccharide (LPS) is an essential and characteristic outer binds to epithelial carcinoembryonic antigen-related cell adhesion membrane component of H. influenzae, also known as lipooligosaccharide molecules.46,48,50

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In addition to promoting binding to the extracellular matrix, PE also Advances in immunisation against the captures vitronectin on the surface of NTHi which protects the bacterial pneumococcus and future strategies pathogen against the lytic activity of the membrane attack complex of the Although considerable progress has been made in the field of complement system.48 Like the pneumococcus, NTHi also protects itself against attack by the alternative and classical pathways of complement immunisation against pneumococcal diseases, particularly the successful activation by binding factor H and C4BP, respectively.50 development of conjugate vaccines which effectively reduce colonisation and disease caused by vaccine-type strains, serotype restriction remains The aforementioned virulence factors of NTHi are summarised in Table 2. a major limitation of current vaccines. Moreover, recent studies have Table 2: Key non-protein and protein virulence factors of NTHi identified the existence of antibody-independent, cell-mediated immune responses triggered by pneumococcal protein antigens, which contribute Factor Activity to clearance of colonisation and possibly prevention of mucosal disease, Lipopolysaccharride Epithelial adhesin, interference with complement clearly highlighting an additional limitation of current vaccines, none of activation, impairs mucociliary escalator function. which contains pneumococcal protein antigens.31 P2, P5 Promotes binding to mucus, adhesion to epithelium (P5). Accordingly, future vaccines will focus on the utilisation of highly- Protein D Impairs mucociliary escalator functions. conserved, broadly serotype-unrestricted, recombinant, surface and HMW1, HMW2 Epithelial adhesin. sub-surface pneumococcal protein antigens. Ideally, these should evoke Hia Epithelial adhesin. the production of both neutralising antibodies and cell-mediated immune

Pili, Hap, Protein E Promotes binding to the extracellular matrix, protects responses. Prominent among the protein antigen vaccine candidates are against the lytic action of complement (PE). PsaA, PspA, Pspc/CbpA, and immunogenic pneumolysoid, a modified pneumolysin devoid of pore-forming activity.12,33,51,52 There are several Host defences operative against NTHi possible types of protein-based vaccines. These are: i) those which contain a single protein; ii) those which contain a cocktail of different As with the pneumococcus, entry of NTHi into the nasopharynx has proteins; iii) a conjugate vaccine in which a single protein is used as a several possible outcomes: i) eradication by innate/adaptive host carrier for pneumococcal capsular polysaccharides; and iv) a conjugate defences; ii) a carrier state in which the bacteria and host co-exist in vaccine in which a cocktail of proteins is used as a carrier system for relative harmony; iii) a state of persistence in which the bacteria are pneumococcal polysaccharides. encased in biofilm either extracellularly, or intracellularly following invasion of epithelial cells, enabling evasion of host defences (this could NTHi-targeted vaccines also be considered as being a chronic infection); and iv) acute infection. In the case of the carrier and persistence states, the bacterial pathogen, Heterogeneity of several major surface antigens, genetic heterogeneity seemingly subdued, may strike when host defences are transiently of strains, and identification of conserved immunogens which evoke compromised, as may occur during infection with influenza or other broadly protective immune responses, have been the major problems respiratory viruses. confronting NTHi vaccine development. Protein D has been identified as a promising vaccine candidate antigen in animal models of experimental In addition, specific mucosal and circulating IgA and IgG antibodies to infection. In a clinical trial involving children in which PD was used as surface proteins of NTHi, innate host defences including the mucociliary an immunogenic carrier protein for pneumococcal polysaccharides escalator, antimicrobial proteins in epithelial lining fluid, TLR/Nod- activated inflammatory responses, and activation of the alternative (11-valent investigational vaccine), significant protection against acute 2 complement pathway are all involved in controlling colonisation. In the otitis media caused by both pneumoccoci and NTHi was achieved. 53 case of TLR-mediated inflammatory responses, OMP-2 and OMP-6 bind Several other vaccines for NTHi are currently in development. to TLR-2, while LPS interacts with TLR-4, resulting in transcription factor Conclusions activation and initiation of an inflammatory response.46 Recognition of LPS is mediated by its receptor, CD14, on cells of the myelomonocytic Ideally, the pneumococcus and NTHi should be eradicated promptly lineage, with the CD14/LPS complex being the apparent ligand for from the respiratory tract to prevent them from taking refuge in TLR-4. An additional protein, MD-2, is associated with TLR-4 and biofilm. Concealment in biofilm provides an environment which may be is necessary for the correct positioning of TLR-4 and its interaction conducive to genetic adaptation and increased virulence from which with LPS. Intra-epithelial sensing of NTHi proteoglycan by Nod 1 also these microbial pathogens can re-emerge when host defences are initiates the production of proinflammatory cytokines. In addition to the transiently compromised. In the case of the pneumococcus, existing aforementioned innate host defences, LPS is also a potent activator of the conjugate vaccines, while clearly effective, may be optimised by vaccine alternative pathway of complement activation, resulting in the production design strategies which incorporate conserved protein antigens which of complement-derived mediators of inflammation, opsonophagocytosis, evoke both antibody and cell-mediated immune responses. Identification and antimicrobial activity. of genetically stable conserved immunogens which evoke broadly protective immune responses is clearly a priority in the development of effective NTHi vaccines.

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