Development of a new tuberculosis vaccine: is there value in the mucosal approach?

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Development of a new tuberculosis vaccine: is there value in the mucosal approach?

TB is a global health problem, killing 1.5 million people every year. The only currently available vaccine, Mycobacterium bovis BCG, is effective against severe childhood forms, but it demonstrates a variable efficacy against the pulmonary form of TB in adults. Many of these adult TB cases result from the reactivation of an initially controlled, latent Mycobacterium tuberculosis infection. Effective prophylactic vaccination remains the key long-term strategy for combating TB. Continued belief in reaching this goal requires unrelenting innovation in the formulation and delivery of candidate vaccines. It is also based on the assumption, that the failure of recent human vaccine trials could have been due to a suboptimal vaccine design and delivery, and therefore should not erode the key principle that a TB vaccine is an attainable target. This report gives a brief overview of the mucosal immune system in the context of M. tuberculosis infection, and focuses on the most recent advances in the field of mucosal TB vaccine development, with a specific emphasis on subunit TB vaccines.

Gil Reynolds Diogo1 & Rajko Reljic*,1 St George’s Hospital, Institute of Infection & Immunity, St George’s University of London, London, SW17 0RE, UK *Author for correspondence: Tel.: +44 208 725 5691 rreljic@ sgul.ac.uk 1

Keywords: adjuvants • BCG • immunity • lungs • mucosal • tuberculosis • vaccine

The threat of TB TB is a major health threat, even more so with the emergence of multidrug-resistant TB (MDR-TB). A third of the world’s population has been infected with Mycobacterium tuberculosis (MTB) [1] . In 2011, the WHO reported 8.8 million incident cases of TB with 1.45 million recorded deaths worldwide [2] . The disease is most commonly transmitted through the inhalation of airborne bacteria contained in droplets, which evade the physical barriers of the respiratory tract reaching the lungs. The treatment of TB comprises a multidrug combination therapy lasting a minimum of 6 months [3] . This protracted treatment is associated with significant side effects and liver toxicity, and though it has somewhat diminished the burden of disease [4] , it frequently leads to patient noncompliance, resulting in relapses or the emergence of drug-resistant strains [5] . Although the TB treatment is still essential to saving lives, it has failed to halt disease progression on the global scale, and mor-

10.2217/IMT.14.62 © 2014 Future Medicine Ltd

bidity and mortality associated with the disease are still very high, highlighting the need for better preventive strategies. Historically, the most efficient way of eradicating a disease has been through the use of a vaccine [6] . However, despite the BCG being used on a large scale since 1974, with over 4 billion doses administered [7] , it has not been able to effectively control TB. The BCG has a remarkable safety record in non immunocompromised individuals and it protects young children against the most dangerous forms of TB. Unfortunately, the protection levels against adult onset of TB are very variable and frequently unsatisfactory, especially in TB endemic areas [7] . It is therefore an imperative to investigate novel ways of improving protection against MTB, which can only be accomplished by better understanding of the pathogen–host interactions. This review will therefore focus on the current understanding of such interactions and the host immune response, and how they could be harnessed to rationally design vaccines against mucosal pathogens such as MTB.

Immunotherapy (2014) 6(9), 1001–1013

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Review Diogo & Reljic Mucosal immune system The immune system includes two major compartments, namely the systemic and the mucosal immune systems [8] . The mucosal surfaces are the first point of contact for many pathogens and therefore are the port of entry. Over 90% of infections occur at the mucosal level, which is no surprise considering the fact that it covers an area of approximately 400 m2 [9] , as opposed to a mere 2 m2 covered by the skin [10] . For this reason, nearly 80% of all immune cells are localized in the mucosa [9] . Epithelial cells line up all mucosal surfaces. The conformation of the epithelia depends on the location in the body and the primary functions, but the basic structure is that of a single or stratified epithelial layer underneath which there is lamina propria, composed of loose connective tissue, and then the region denominated as submucosa [10] . The epithelial lining differs from site to site; stratified epithelium is found in the oral cavity, esophagus and lower genital tract, while simple epithelium lines the airways, gastrointestinal tract and upper genital tract [10] . The concept of common mucosal system can be explained by homing mechanisms. Immune effector cells in the mucosa possess characteristic homing mechanisms enabling them to migrate and return to the appropriate mucosal sites [11] . Induction of an immune response at a single mucosal tissue may lead to the widespread IgA production at different mucosal surfaces [12] . This phenomenon is due to the fact that mucosal lymphoid tissues possess homing receptors for chemokines and adhesion molecules that identify complementary molecules in the endothelium of the mucosa [13,14] . IgA producing B cells activated at mucosal sites possess the CCR10 receptor that recognizes CCL28 released by epithelia across mucosal sites including the respiratory tract [12] . MTB enters the body through the airways and the host’s response is the result of cooperation between the innate and acquired immune systems. The initial step is the recognition of the pathogen followed by the activation of the innate response and subsequent recruitment of the acquired immunity [15] . Upon entering the lungs by aerosols, the pathogen encounters macrophages and dendritic cells (DCs). Some of the bacilli are destroyed but others endure and replicate within the macrophages. DCs and activated macrophages produce immune effectors such as IL-12 and IL-18 that induce natural killer (NK) and T cells which in turn secrete IFN-γ. The latter cytokine plays a major role in immunity to MTB, primarily through activation of macrophages and their bacterial killing by multiple mechanisms. Macrophages One of the first cells to interact with the pathogen is the alveolar macrophage [16] . The immune response to

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MTB is activated through the detection of pathogenassociated molecular patterns by the host’s germline encoded pattern recognition receptors [17] . The principal pattern recognition receptors involved in the recognition of MTB are the Toll-like receptors (TLRs). Lipoarabinomannan, a key constituent of the pathogen cell wall, is also recognized by phagocytic cells. Macrophages phagocytose the invading bacteria triggering a proinflammatory response. This may lead to the typical granuloma formation. Macrophages are capable of bringing about MTB destruction mediated by the phagosome lysosome fusion, the action of specific defensins, proteases, iron and sidephore-binding molecules [18] . Upon TLR activation MTB killing can also be mediated by reactive oxygen intermediates or reactive nitrogen intermediates [18] . Macrophages can also present antigens to T cells and play an important role in early stages of induction of acquired immune response. DCs Of crucial importance to sampling antigens in mucosal sites is the ability of DCs to squeeze their dendrites between the junctions of the epithelial cells and sample the outer lumen directly [19] . Once the DCs have been primed, they can activate surrounding memory lympho cytes eliciting either memory [20] recall or tolerance [21] . The DCs can also migrate through the lymphatic system to the lymph nodes to stimulate naive T cells [8] . DCs are the principal APC of the immune system. They can be classified as tissue DCs found in the peripheral organs such as the mucosa, skin and internal organs or blood DCs found in circulation [22] . In the respiratory tract, DCs occupy prime positions for antigen sampling and are located in the epithelium and beneath in the lamina propria, in the lung parenchyma and alveolar gaps [23,24] . In the context of MTB infection, DCs can phagocytize bacteria [25,26] and thus play a major role in activating T cells. They are also capable of secreting cytokines themselves, including IFN-γ [27] . DCs act as a single cell bridge between the innate and acquired immunity. Upon internalization of MTB and cell activation by the TLRs, they migrate to the draining lymph nodes, where they mature and present the processed MTB antigens to the naive T cells triggering the acquired immunity [16] . Antigen sampling The mucosal tissues possess inductive sites that play a role in antigen sampling, and subsequent induction of naive T and B cells prior to their localization to the effector sites [28] . Antigen sampling and processing at the mucosal level is performed by DCs and specific microfold (M) cells, localized on the surface

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of the mucosal-associated lymphoid tissues and Peyer’s patches [29] . Peyer’s patches are the fundamental inductive sites possessing a large arsenal of immune cells. The sampling of antigens varies depending on the particular nature of the mucosal site and corresponding epithelial lining, but all rely on DCs [30] . The DCs potentiate the immune response and activate the acquired immunity [31] . M cells outline intraepithelial compartments to where lymphocytes and DCs localize. These highly dedicated epithelial cells then convey the antigens through vesicular transport from the lumen to DCs situated in the adjacent pocket [10] . Antigen sampling by M cells is normally linked to the induction of secretory IgA [32] . The M cells are readily found in the lymphoid tissues associated with the digestive system but they can also be found in the respiratory mucosa where they contribute to antigen sampling of the respiratory pathogens [33] . For example, the analysis of nasal airway passages revealed the presence of M cells that were able to take up ovalbumin and recombinant Salmonella typhimurium expressing green fluorescent protein [34] . Moreover, these M cells were also capable of taking up respiratory pathogen group A Streptococcus after nasal challenge. MHC class II peptides of the MTB must be processed by professional APC so they can be presented to CD4 + T cells [35] . On the other hand, MHC Class I molecules, found on all cells apart from erythrocytes, are capable of presenting antigens to specific CD8 + T cells and they are responsible for killing of infected cells that harbor bacteria in the cytosol [36] . Both CD4 + and CD8 + T cells are thought to be essential for conferring protection against MTB infection. Alongside alveolar macrophages and DCs, they are the principal immune effector cells in the context of a MTB infection. Schematic representation of a mucosal immune response in MTB infection is depicted in Figure 1. Humoral responses The general consensus is that the extent of protection against MTB infection relies primarily on an efficient cellular immunity. However, more recently evidence has emerged regarding the protective role of antibodies against MTB [37–40] . This emerging evidence suggests that B cells and humoral immunity can modulate the immune response to various intracellular pathogens, including MTB. Thus, the B lymphocytes appear to form aggregates in the lungs of TB patients, nonhuman primates and mice, which display features of germinal centers [41] . A potent indicator of a strong mucosal immune response is the release of secretory IgA [31] . IgA can form dimers and the higher polymers, which can translocate


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through the epithelia upon binding to polymeric Ig receptor, eventually accumulating on the apical side as the secretory IgA (sIgA). sIgA plays a major role in immune exclusion and other immunity functions at the mucosal sites [31] . There is also a role for IgG at the mucosal sites; it normally locates to these sites by passive diffusion from the blood but can sometimes result from localized secretion. IgG is often associated with immunopathology at mucosal sites due to its proinflammatory capabilities [31] . IgA plays a crucial role in antimicrobial functions. In addition to pathogen neutralization intracellularly during translocation [42] , sIgA can also agglutinate microbes, facilitating their clearance by peristaltic and ciliary mechanisms [43] . Furthermore, sIgA can impede the entry of pathogens (blocking effect) by forming a complex with bacterial cell wall components involved in bacterial–host cell interactions. It appears that IL-17 may play an important role in propagation and differentiation of B-cells producing sIgA [44] . Using an IgA monoclonal antibody, we have shown that it is possible to confer a degree of protection against MTB in mice by intranasal inoculation of the antibody, especially when co-administered with immunopotentiating cytokine IFN-γ [45,46] . Cellular responses Despite the important role of sIgA-producing B cells, the principal immune effector cells at the mucosal level are T lymphocytes. It is thought that T cells make up 80% of the entire lymphoid cell count at the mucosal sites [47] . In the context of MTB, CD4 + T cells are the main producers of IFN-γ that activates macrophages [48] , which is critical for controlling and eliminating MTB. The CD4 + T-cell responses are initiated in the lungdraining and mediastinal lymph nodes [49,50] . Following aerosol challenge with MTB, highly activated CD4 + T cells could be recovered from the lungs [51] and similarly, vaccinated mice also display abundant antigen-specific CD4 + T cells in their lungs [52] . However, increasing evidence suggests that effector CD4 + T-cell responses to MTB during an acute infection are delayed [49,53] , which may be the main reason for the failure of the immune system to eliminate the pathogen, and instead, allow it to establish a persistent infection. CD8 + T cells are also thought to be important in mounting a successful immune response to MTB. They have been shown to be involved in the lysis and apoptosis of infected cells and subsequent killing of intracellular MTB [54] . Therefore, both CD4 + and CD8 + T cells play a crucial role in mucosal immunity to pathogens and this will be further elaborated in the context of various vaccination strategies for TB described below.

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sIg A

Alveolar macrophage

MTB Antigens Mucus

M cell

Epithelial cells Mucosal homing DC Lymphocytes

Draining lymph node B cell

Systemic immunity

Naive lymphocyte

T cells

Figure 1. Antigen sampling at a mucosal site. The antigen can be translocated through the M cells to the underlying immune cells or can be sampled directly by the DCs that extend between adjacent epithelial cells. This partially mimics the way that Mycobacterium tuberculosis gains access to the basolateral side of the alveolar epithelium. The DCs then migrate to the draining lymph nodes where they prime naive lymphocytes. The immune response is then propagated back to the mucosal surface by means of the homing mechanisms and is also spread systemically. DC: Dendritic cell; sIg A: Secretory IgA.

However, in the context of mucosal vaccination, an important issue concerning induction of effective CD4 + and CD8 + T-cell responses is to circumvent the immune tolerance. The systemic immune cells operate naturally in a relatively sterile environment. On the other hand, the mucosal immune system is persistently stimulated by foreign antigens, be it food associated antigens in the gut or airborne particles in the airways. Consequently, it is tightly controlled by a suppressive mechanism [55] to avoid constant inflammation and hypersensitivity [56] . While the exposure of epithelial mucosa to soluble antigens sometimes induces tolerance through immunosuppression in a bid to reach homeostasis, this regulatory mechanism appears to be less significant in the airways as opposed to the gastric system or responses toward systemically presented antigens [31,57] . Therefore, respiratory route

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of delivery lends itself well for vaccination against airway-associated pathogens such as MTB. Mucosal vaccines Historically, early mucosal vaccines were based on whole, usually attenuated or sometimes killed organisms, delivered orally. One of the pioneering mucosal vaccines was tested in 1922 and was based on killed pneumococcal bacteria that were used to immunize rabbits, resulting in a very robust protection against the pathogenic challenge [58,59] . Only a relatively low number of these early mucosal live attenuated mucosal vaccines have reached the market owing to the fear and potential dangers of these types of vaccines when weighed up against the benefits, especially in the context of rare diseases [31] . Licensed mucosal vaccines cover a range of enteric and respiratory pathogens



as well as the mucosally transmitted neurological pathogen, the poliovirus. It has been shown that mucosal vaccines, especially those delivered intranasally, could generate high IgA and IgG titres not only in the mucosa but also in the serum [60,61] . On the other hand, systemic vaccines often fail to recruit immune responses to the mucosal surfaces. This can be explained by the fact that antigens are sampled by DCs away from the mucosal surfaces and so, the mucosal homing mechanisms are not activated [62] . In comparison, mucosal vaccination leads to both mucosal and systemic responses due to DCs carrying the sampled antigen to systemic inductive sites such as the spleen and lymph nodes [63,64] . Likewise, some B cells that may be activated at the site of mucosal antigen contact express the systemic homing receptors α4/β1 and l-selectin [14] and can enter circulation. An attractive feature of mucosal vaccines is that they are easier to use compared with their needle-dependent counterparts. An array of delivery systems have been proposed, comprising liquid injectors, transdermal application to the skin or mucosa, sublingual administration and orally administered or nasal sprays [65] . For a vaccine to be successful against a mucosal pathogen, it needs to induce robust protective responses locally but also prevent systemic dissemination [10] . A number of mucosally targeted vaccines against some of the biggest human pathogens are currently being researched, including diseases such as TB [66,67] and HIV [68] . Some are at clinical trial stages. In addition, vaccines against the enteric diseases such as enterotoxigenic Escherchia coli (ETEC) [69] and Shigella [70] have been refined but are yet to be approved. Mucosal vaccines for TB As MTB primarily targets the airways, mucosal immunization via the respiratory route has received considerable attention. Mucosal immunization targeting the airways has several practical advantages, including the ease of delivery, absence of pain and fear associated with use of needles and reduced risk of pathogens transmission associated with contaminated needles. More importantly, as mentioned above, mucosal immunizations have been shown to induce both strong mucosal and systemic immunity [71] . Despite the fact that MTB is a mucosal pathogen that targets primarily the lungs, the only licensed vaccine, the BCG, is delivered intradermally. Although it confers reasonable protection against severe forms of primary MTB infection in children, it has many limitations including failure to prevent the onset of adulthood TB, with the duration of protection being estimated at 15 years [72] . Being a live attenuated vac-


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cine, it is contraindicated in immunocompromised individuals [9] , in particular HIV positive patients who are particularly susceptible to TB and have contributed to the surge of TB globally in the recent past [73,74] . It is widely agreed that the mucosal immune response is vital in protecting against TB and yet, the vast majority of new vaccine candidates are systemically administered. They typically induce strong systemic but weak mucosal immune responses and this may not be enough to confer a long-lasting protection. On the other hand, some of the current mucosal approaches have shown a considerable promise, as summarized in Table 1. The site of challenge should be taken into account when choosing administration route for mucosal vaccines. Nasal or sublingual vaccines have been shown to be the most powerful of mucosal vaccines at inducing systemic responses, sometimes even being comparable to systemic vaccination [10,64] . There is an increasing body of evidence that mucosal immunizations induce a more protective immunity against mucosal pathogens than the standard parenteral vaccines [47] . Intranasal immunizations are particularly effective against respiratory pathogens such as influenza and MTB. To effectively induce cellular and humoral responses at mucosal as well as systemic levels some approaches have investigated the added value of heterologous vaccine regimens with boost immunizations that could cover both systemic and mucosal delivery. To best induce the desired immune response, efforts should be made for these mucosal vaccines to mimic the pathogen itself [8] . There is an obvious need to increase the robustness of protection conferred by TB vaccines. An array of different techniques are currently being explored ranging from recombinant strains of BCG, a selection of MTB subunit antigens as well as enhancing and honing of immunity with adjuvants or novel delivery systems such as nanoparticles. Recombinant viral vector vaccines The principle behind this approach is the use of modified viruses that contain genetic material coding for a particular antigen of interest. Wang et al. showed that immunizing mice mucosally with a recombinant adenovirus based vaccine candidate against TB expressing Ag85A conferred more robust protection than parenteral immunization [77] . Intranasal immunization elicited higher immune responses at the mucosal sites compared with parenteral vaccination that generated higher systemic responses, in particular CD8 + T cells in the peripheral lymph nodes, but not in the lumen of the airways. A one off intramuscular immunization with AdAg85A conferred minimal protection to mice challenged with MTB but the little protection it did confer


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Table 1. Examples of mucosally targeted vaccines against tuberculosis. Vaccine

Prime

Route

Delivery/adjuvant

rhPIV2-Ag85B

None

Intranasal

Viral vector– CFU reduction in lungs and spleens. recombinant ParaPreferential immune response to Ag85B influenza type 2 virus over viral vector. Mice

[75]

MV85A

BCG prime

Intranasal

Viral vector–modified Tolerable. Highly immunogenic. No Vaccinia Virus Ankara detectable serum antivector antibody response contrary to the intradermal version. Nonhuman primates

[76]

AdAg85A

None or BCG prime

Intranasal

Viral vector– replication deficient adenovirus

A single intranasal but not intramuscular immunization provided robust protection, greater than BCG. High retention of antigen specific T cells

[77,78]

Ad35/ AERAS-402

BCG prime

Intranasal

Viral vector– replication deficient adenovirus

Significant enhancement of BCGinduced immunity in mice; also shown to induce polyfunctional CD4 T cells and strong CD8 T-cell responses in humans that were 50-fold higher than those detectable preboost

[78,79]

Nano-AH (Ag85BHBHA)

BCG prime

Intranasal

YC-NaMA nanoparticles plus fusion protein

Reduced bacterial load in lungs and spleens. Robust humoral and cellular responses. Polyfunctional CD4 + T cells expressing IFN-γ, IL-2 and TNF-α. Mice

[80]

Ag85B/CpG

BCG prime

Intranasal

CpG

Reduced BCG load after challenge but lung infection was not prevented. Increased airway DCs detected early post challenge correlated with higher protection. CD4 + Th1 and CD8 + cells detected in airways. Mice

[66]

HU58k-rAcr-Ag85B BCG prime and Intranasal and HU58kno prime rMPT64

HU58k spore

Antigen specific lung sIgA was detectable in the lung lavage. Evidence of systemic T-cell proliferation, IFN-γ production and CFU reduction in the lungs. Mice

[81]

TB-RICs

BCG prime

Plant expressed Antibody and cellular immune responses recombinant immune and a statistically significant protection complexes in mice when used to boost BCG. Mice

[82]

ESAT-6-Ag85BMPLSE

ESAT-6-Ag85B- Oral DDA-MPL

Stable emulsified detoxified monophospholipid A (MPL)

IFN-γ production in the spleen and CFU reduction. Mice and guinea pigs

[83]

H-kBCGEurocrine™ L3

H-kBCGNasal Eurocrine™ L3

Eurocrine™ L3

The intranasal administration gave equal or better levels of protection than subcutaneous. High-level immune responses in particular serum IgG and IFN-γ. A tendency toward a Th2 bias

[84]

Intranasal

Study outcomes. Infection model

Ref.

CFU: Colony forming units; DC: Dendritic cell; HBHA: Hemagglutinin adhesion protein; MPL: Monophospholipid; TB-RIC: Tuberculosis-recombinant immune complex.

was merely transient and mice were no longer protected after 4 weeks [77] . On the other hand, intranasal immunization with AdAg85A conferred long-term robust protection against pulmonary TB and systemic disease dissemination better than the subcutaneous BCG

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immunization [77] . Another study based on AdAg85A vaccine demonstrated that intramuscular immunization strongly induced CD8 + T cells systemically, but not in the airway lumen analyzed [85] . Even though some CD8 + T cells could be located in the lung interstitium



following intramuscular immunization, they did not efficiently migrate to the lumen post-challenge, thus conferring little or no protection. In contrast, intranasal immunization induced much lower levels of T-cell activation in the spleen but both CD4 + and CD8 + T cells were robustly activated in the lung and particularly in the lumen of the airways. Thus, intranasal immunization, selectively induces and retains T cells in the lung interstitium and airway lumen, providing robust protection, in keeping with the fact the lungs are the primary site of MTB entry [85,86] . Further examples of vaccines from this category are the Crucell Ad35/AERAS-402 and the MVA-Ag85A vaccines. Ad35/AERAS-402 is an Ad35 vectored TB vaccine candidate being developed by Crucell, a leading biotech in Europe, and Aeras. It contains three MTB antigens, antigens 85A, 85B and TB10.4 [78] . It is currently in Phase 2b clinical trial. The MVA-85A vectored subunit vaccine, based on recombinant, replication deficient, modified Vaccinia Virus Ankara (MVA) expressing Ag85A [87,88] recently completed Phase IIb clinical trials but failed to confer added protection to the BCG-immunized subjects [89] . Inactivated or killed vaccines These are composed of viral constituents or bacteria that are purposely cultured and then inactivated by autoclaving, γ-rays or treatment with formaldehyde. Examples of some of these licensed vaccines are Salk polio vaccine, influenza vaccine, vicapsular polysaccharide typhim Vi® vaccine, Sanofi Pasteur; Typherix® and GSK typhoid fever vaccines and the cholera Dukoral® Crucel vaccine [10] . Mice immunized intranasally with killed BCG adjuvanted with a mutant Escherichia coli enterotoxin produced a characteristic Th1 responses and the bacterial load was reduced after BCG infection. Although it is reported that Ag85B and other important antigens are downregulated in killed BCG, low-level responses to this antigen were reported and a way of further enhancing immune responses could be to perhaps add potentially important immunogenic subunits to such vaccine formulations [90–92] . Live attenuated vaccines Live attenuated immunization approaches are based on compromised nonvirulent variants of the pathogen itself. Historically, this class of vaccines are very successful and count among their ranks many viral vaccines such as measles, mumps rubella, chicken pox, oral polio the Sabin vaccine, yellow fever, intranasal influenza, rabies and also bacterial vaccines such as BCG and typhoid [10] . A disadvantage of non replicating vaccines is that they are more likely to require multiple doses to achieve the desired potency of immune


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response whereas live vaccines offer a more continuous supply of antigen [10] . A recombinant attenuated MTB strain with mutations in the replication and immune evasion genes and expressing SIV genes was administered mucosally to nonhuman primates, was shown to induce persistent vaccine-specific antibody responses, with both SIV and TB-specific CD4 + and CD8 + T cell cells at a mucosal and systemic level. It was also shown to be safe in SIV-infected infant animals [67] . Live attenuated vaccines tend to show a strong correlation between effector immune responses and protection [11] and this has been attributed to the array of antigens displaying their immunogenic properties. This is not surprising in view that these types of vaccines resemble most closely the actual pathogens and therefore, induce strong humoral and cellular responses with lasting memory [10] . There is, however, a drawback for live attenuated vaccine candidates in the context of TB, since targeting the lungs would require aerosol or nasal administration and therefore the inactivated pathogen would be proximal to the blood–brain barrier. Live attenuated vaccines are associated with the risk of return to virulence, which is a major safety issue, as was the case for the oral polio vaccine Sabin 3 [93] . Therefore, safer alternatives for mucosal TB vaccines are preferred. One such alternative is the subunit vaccines, not requiring live attenuated microorganisms. Subunit vaccines Subunit vaccines rely on proteins or epitopes common to the pathogen itself to trigger the required immune response. Subunit vaccines can be composed of recombinant proteins alone, combined with adjuvants or expressed by attenuated viral vectors [94] . They can be based on a single epitope or a combination, the latter often being more effective [95] . The combination subunit vaccines can be administered as a mix of proteins or as a fusion protein selecting a number of desired proteins or peptides. The fusion protein approach allows for greater reproducibility and also simplifies purification steps [95] . These vaccines rely on selecting an immunogenic antigenic component of the pathogen, be it a protein alone or linked to a polysaccharide. Currently the only licensed mucosal subunit vaccine is the cholera vaccine. This vaccine is composed of cholera toxin B subunit coupled with the inactivated pathogen. Oral immunization with Dukoral vaccine conferred protective, lasting immunity but parenteral immunization did not [96] . A nasal subunit vaccine candidate against hepatitis B has been tested in a Phase I clinical trial and was found to be safe, well tolerated and immunogenic in healthy adults [97] .


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Review Diogo & Reljic Many antigen-based subunit vaccines for TB have been tested, including antigens such as Ag85A,B, ESAT-6, CFP-10, MPT64, MPT83, Hsp65 and KATG. Due to the relative efficacy of BCG in infants, and the implications of implementing a clinical trial where certain subjects (i.e., those already BCG- vaccinated) would have to be excluded, a subunit vaccine would more than likely be applied as a heterologous boost approach. Several types of adjuvants and delivery systems have been proposed for mucosal delivery but caution needs to be exercised when considering their application in clinical studies, as a good safety profile obtained from testing in animals may not necessarily translate to humans. Particles To achieve an optimal vaccine delivery, many different approaches are being contemplated for the subunit vaccines. This ranges from formulating antigens with adjuvants, to the use of carriers such as liposomes or particles. Nanocapsules and nanoparticles have been extensively explored for their adjuvantic properties and as antigen carriers. They offer a platform to present one or more antigens on their surface or incorporated in the lipid membrane, thus partially mimicking pathogen itself. The main three types of nanocapsules considered for mucosal administration are the water in oil emulsion, oil in water emulsion and liposomes [6] . Nanoparticles can range from 20 to 500 nm in size and can be used as a deposit for slow release of antigen over time. They can be of many different origins and typically capture antigens on their surface or in their matrix [6] . A significant challenge encountered by all mucosal immunization approaches is overcoming the mucosal tolerance mechanisms, whereby the mucosal sites inhibit immune and inflammatory responses to commensal organisms and food and environmental antigens that it may encounter in order to avoid unnecessary activation. To circumvent this issue, adjuvants can be used to further enhance the immune responses elicited by mucosal vaccines. An oral vaccine coupled with pH-dependent microparticles allowing for large intestine targeted delivery was capable of inducing immune responses in mice comparable to rectal delivery and was able to confer protection against vaginal and rectal viral challenge [98] . Antigens enveloped in biodegradable microparticles delivered intranasally have been administered to mice, inducing wide-range immune responses [99,100] . Recently, pluronic-stabilized polypropylene sulphide nanoparticles have been used as a delivery platform for TB vaccine candidates [66] . These nanoparticles are approximately 30 nm in size and can activate the

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complement cascade because of the chemistry of their surface. The highly immunogenic Ag85B antigen was used alongside the CpG adjuvant and delivered to the lungs and intradermally. Mucosal delivery of the nanoparticles, antigen and adjuvant combination induced superior antigen specific polyfunctional Th1 responses in the spleen, the lung draining lymph nodes and the lung itself, and conferred greater protection compared with the soluble antigen and adjuvant alone, or intradermally administered vaccines. In another study, ESAT-6 encapsulated in poly(lactide) microspheres administered intranasally to mice induced higher level of ESAT-6 specific IFN-γ secreting cells compared with those after intramuscular immunization [101] . Most recently, we have shown that mucosal boosting of BCG primed mice with the nano-AH vaccine candidate composed of Ag85B and heparin-binding hemagglutinin adhesion protein (HBHA), formulated with nanoparticles derived from the wax of the yellow carnauba palm tree, induced robust humoral and cellular responses and multifunctional T cells expressing IFN-γ, IL-2 and TNF-α. Importantly, this immunization regimen conferred a significant further protection against MTB infection compared with BCG alone [80] . In a similar mucosal approach, we also showed that inactivated Bacillus subtillis spores coated with a MPT64 or Ag85B-Acr antigens induced protective immunity in intranasally immunized mice, although this vaccine appeared to perform better in the homologous than in the heterologous (BCG boost) immunization regimen [81] . In both these instances, nanoparticles and the bacterial spores acted as antigen delivery vehicles and adjuvants, demonstrating that particles have the potential to induce appropriate mucosal and systemic immune responses. Adjuvants There is a wide consensus that for a mucosal vaccine to be successful the antigen must be successfully delivered to the immune induction sites and therefore the route of immunization must be chosen accordingly. Thus for MTB, lung targeting is the preferable mode of antigen delivery, as this is the primary site of infection. Live attenuated vaccines and replication-deficient vectors are the most efficient and robust way of inducing mucosal immunity but caution must be exercised with regard to their safety and the potential risk of lung immunopathology. In addition, whole organisms such as recombinant BCG, though carrying significant promise as BCG-replacement systemic vaccine, may not be suitable for intranasal delivery due to the proximity of the brain and potential damage to the blood–brain barrier. Protein subunit vaccines are a highly attractive alternative option for mucosal antigen delivery against



TB and also other infections, but unfortunately, the immune responses they induce are often not robust enough, and consequently, various modulators or adjuvants must be used. There is a need to develop suitable mucosal adjuvants and many different approaches have been tested and shown to carry significant vaccine potential. Thus, a large number of immunomodulators and adjuvants are currently being considered, including aluminium salts, oil in water emulsions [102] , DNA [103] , lipid A [104] , cholera enterotoxin [103] , E. coli heat labile enterotoxin [105] , emulsions and particles [106] , eurocrine L3 [84,107] and cytokines [108] . However, we demonstrated recently that it is possible to circumvent the need for exogenous adjuvants altogether and instead design a vaccine with a built-in molecular adjuvanticity. Thus, we recently showed [82] that recombinant immune complexes (RICs) based on Ag85B-Acr fusion protein and a monoclonal antibody against Acr, and expressed as self-polymerizing complexes in transgenic tobacco plants, could induce protective immune responses in mice after intra nasal immunization. These TB-RICs targeted the Fc immunoglobulin receptors on APC and could activate complement, which was the basis of their adjuvanticity in vivo, and protection against MTB infection. Such novel, highly innovative approaches require further testing, so that, together with the more conventional mucosal adjuvants and delivery systems, these studies may hopefully translate into an effective mucosal immunization strategy against TB in the near future.

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TB, a more robust and reliable vaccine is needed to eradicate this disease. Ideally, such vaccine should be given mucosally, and should be able to boost BCG, considering that the latter is still widely administered around the world and protects against the most severe forms of primary TB disease in young children. Historically, live attenuated vaccines have given the best results against many infections, but there are many safety concerns regarding their mucosal use, especially in the context of such a deadly pathogen as MTB, and also in the view that a significant proportion of intended vaccine targets are immunocompromised individuals. To circumvent this issue, subunit vaccines are being explored as safer alternatives, but for this approach to succeed, highly innovative delivery systems and adjuvants are needed, to overcome mucosal tolerance and induce protective anti-TB immunity. When applied as a boost to BCG, such mucosal TB vaccine that stimulates and perpetuates mucosal immunity may not only impede the initial infection at the airways but may also confer a better protection against reactivation TB, which is the biggest challenge for the new vaccine. The research over the last several years is encouraging and holds promise that such a vaccine may be feasible and should be pursued with renewed vigor. Acknowledgements The authors would like to thank M Paul and P Martins for assistance with the graphics.

Financial & competing interests disclosure

Future perspective The existing licensed vaccines have allowed for a great reduction in the mortality and morbidity of diseases caused by enteric pathogens such as rotavirus, Vibrio cholera and Salmonella typhi as well as mucosally targeted pathogens that disseminate to the distal organs, such as poliovirus. Although BCG has made a positive impact on limiting the global burden of

R Reljic’s position is funded by Sir Joseph Hotung Foundation. The authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed. No writing assistance was utilized in the production of this manuscript.

Executive summary • An efficient way of developing a mucosal vaccine should combine the knowledge of the pathogenesis with that of the properties of the particular mucosal immune system being targeted. The preferred approach is to mimic the pathogen itself, therefore inducing the appropriate immune response at the correct site. For example, for respiratory pathogens, vaccines need to be targeted to the airway and preferably should adhere or interact with the inner mucosa. • In the context of TB, several mucosal approaches have been considered, including replication-deficient viral vectors, live attenuated organisms and subunit vaccines. • The subunit approach is an attractive alternative to live organisms or recombinant viral vectors but requires suitable adjuvants and delivery systems. Several adjuvants and vaccine delivery platforms have been tested and have shown considerable promise in experimental models of infection. • The challenge now is to further optimize and develop these mucosal approaches in preclinical and clinical trial studies, that may lead to a new BCG-boost TB vaccine.



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Review Diogo & Reljic References 1

Beresford B, Sadoff JC. Update on research and development pipeline: tuberculosis vaccines. Clin. Infect. Dis. 50 (Suppl. 3), S178–S183 (2010).

2

WHO. Tuberculosis Fact Sheet No. 104 (2012). www.who.int/mediacentre/factsheets/fs104/en/

3

Royce S, Grzemska M. Treatment of tuberculosis guidelines (2012). http://whqlibdoc.who.int/ publications/2010/9789241547833_eng.pdf?ua=1

4

5

Smith JP. Nanoparticle delivery of anti-tuberculosis chemotherapy as a potential mediator against drug-resistant tuberculosis. Yale J. Biol. Med. 84(4), 361–369 (2011).

6

Chadwick S, Kriegel C, Amiji M. Nanotechnology solutions for mucosal immunization. Adv. Drug Deliv. Rev. 62(4–5), 394–407 (2010).

7

Kaufmann SHE, Hussey G, Lambert P. New vaccines for tuberculosis. Lancet 375(9731), 2110–2119 (2010).

8

Neutra MR, Kozlowski PA. Mucosal vaccines: the promise and the challenge. Nat. Rev. Immunol. 6(2), 148–158 (2006).

9

Li W, Deng G, Li M, Liu X, Wang Y. Roles of mucosal immunity against Mycobacterium tuberculosis infection. Tuberc. Res. Treatment 2012, 791728 (2012).

10

Kraehenbuhl J, R Neutra M. Mucosal vaccines: where do we stand? Curr. Top. Med. Chem. 13(20), 2609–2628 (2013).

11

Czerkinsky C, Holmgren J. Mucosal delivery routes for optimal immunization: targeting immunity to the right tissues. Curr. Top. Microbiol. Immunol. 354, 1–18 (2012).

Fagarasan S, Kawamoto S, Kanagawa O, Suzuki K Adaptive immune regulation in the gut: T cell-dependent and T cellindependent IgA synthesis. Annu. Rev. Immunol. 28, 243–273 (2009).

21

Mayer L, Shao L. Therapeutic potential of oral tolerance. Nat. Rev. Immunol. 4(6), 407–419 (2004).

22

Itano AA, Jenkins MK. Antigen presentation to naive CD4 T cells in the lymph node. Nat. Immunol. 4(8), 733–739 (2003).

23

Masten BJ, Lipscomb MF. Comparison of lung dendritic cells and B cells in stimulating naive antigen-specific T cells. J. Immunol. 162(3), 1310–1317 (1999).

24

von Garnier C, Filgueira L, Wikstrom M et al. Anatomical location determines the distribution and function of dendritic cells and other APCs in the respiratory tract. J. Immunol. 175(3), 1609–1618 (2005).

25

Bodnar KA, Serbina NV, Flynn JL. Fate of Mycobacterium tuberculosis within murine dendritic cells. Infect. Immun. 69(2), 800–809 (2001).

26

Gonzalez-Juarrero M, Orme IM. Characterization of murine lung dendritic cells infected with Mycobacterium tuberculosis. Infect. Immun. 69(2), 1127–1133 (2001).

27

Giacomini E, Iona E, Ferroni L et al. Infection of human macrophages and dendritic cells with Mycobacterium tuberculosis induces a differential cytokine gene expression that modulates T cell response. J. Immunol. 166(12), 7033–7041 (2001).

28

Tamura S, Kurata T. Defense mechanisms against influenza virus infection in the respiratory tract mucosa. Jpn J. Infect. Dis. 57(6), 236–247 (2004).

29

Kiyono H, Fukuyama S. NALT-versus Peyer’s-patch-mediated mucosal immunity. Nat. Rev. Immunol.4(9), 699–710 (2004).

30

Brandtzaeg P, Farstad IN, Haraldsen G. Regional specialization in the mucosal immune system: primed cells do not always home along the same track. Immunol. Today 20(6), 267–277 (1999).

31

Ogra PL. Mucosal immunity: some historical perspective on host-pathogen interactions and implications for mucosal vaccines. Immunol. Cell Biol. 81(1), 23–33 (2003).

32

Neutra M, Kraehenbuhl J. Cellular and molecular basis for antigen transport across epethilial barriers. In: Mucosal Immunology. Mestecky J, Lamm ME, McGhee JE, Bienenstock J, Mayer L, Strober W (Eds). Academic Press, New York, 111–118 (2005).

12

Woof JM, Mestecky J. Mucosal immunoglobulins. Immunol. Rev. 206(1), 64–82 (2005).

13

Kunkel EJ, Butcher EC. Plasma-cell homing. Nat. Rev. Immunol. 3(10), 822–829 (2003).

14

Sigmundsdottir H, Butcher EC. Environmental cues, dendritic cells and the programming of tissue-selective lymphocyte trafficking. Nat. Immunol. 9(9), 981–987 (2008).

15

Kleinnijenhuis J, Oosting M, Joosten LA, Netea MG, Van Crevel R. Innate immune recognition of Mycobacterium tuberculosis. Clin. Dev. Immunol. 2011, 405310 (2011).

16

Bhatt K, Salgame P. Host innate immune response to Mycobacterium tuberculosis. J. Clin. Immunol. 27(4), 347–362 (2007).

33

Akira S, Tokeda K, Kaisho T. Toll-like receptors: critical proteins linking innate and acquired immunity. Nat. Immunol. 2(8), 675–680 (2001).

Kraehenbuhl J, Neutra MR. Epithelial M cells: differentiation and function. Annu. Rev. Cell Dev. Biol. 16(1), 301–332 (2000).

34

Korbel DS, Schneider BE, Schaible UE. Innate immunity in tuberculosis: myths and truth. Microb. Infect. 10(9), 995–1004 (2008).

Kim DY, Sato A, Fukuyama S et al. The airway antigen sampling system: respiratory M cells as an alternative gateway for inhaled antigens. J. Immunol. 186(7), 4253–4262 (2011).

35

van Crevel R, Ottenhoff TH, van der Meer JW. Innate immunity to Mycobacterium tuberculosis. Clin. Microbiol. Rev. 15(2), 294–309 (2002).

36

Mazzaccaro RJ, Gedde M, Jensen ER et al. Major histocompatibility class I presentation of soluble antigen facilitated by Mycobacterium tuberculosis infection. Proc. Natl Acad. Sci. USA 93(21), 11786–11791 (1996).

17

18

19

1010

Aagaard C, Dietrich J, Doherty M, Andersen P. TB vaccines: current status and future perspectives. Immunol. Cell Biol. 87(4), 279–286 (2009)

20

Rescigno M, Urbano M, Valzasina B et al. Dendritic cells express tight junction proteins and penetrate gut epithelial monolayers to sample bacteria. Nat. Immunol. 2(4), 361–367 (2001).




37

38

39

40

41

42

Costello AM, Kumar A, Narayan V et al. Does antibody to mycobacterial antigens, including lipoarabinomannan, limit dissemination in childhood tuberculosis? Trans. R. Soc. Trop. Med. Hyg. 86(6), 686–692 (1992). Sanchez-Rodriguez C, Estrada-Chavez C, Garcia-Vigil J et al. An IgG antibody response to the antigen 85 complex is associated with good outcome in Mexican Totonaca Indians with pulmonary tuberculosis. Int. J. Tuberc. Lung Dis. 6(8), 706–712 (2002). Vordermeier H, Venkataprasad N, Harris D, Ivanyi J. Increase of tuberculous infection in the organs of B cell‐ deficient mice. Clin. Exp. Immunol. 106(2), 312–316 (1996). Rodríguez A, Tjärnlund A, Ivanji J et al. Role of IgA in the defense against respiratory infections: IgA deficient mice exhibited increased susceptibility to intranasal infection with Mycobacterium bovis BCG. Vaccine 23(20), 2565–2572 (2005). Kozakiewicz L, Phuah J, Flynn J, Chan J. The role of B cells and humoral immunity in Mycobacterium tuberculosis infection. Adv. Exp. Med. Biol. 783, 225–250 (2013). Mazanec MB, Kaetzel CS, Lamm ME, Fletcher D, Nedrud JG. Intracellular neutralization of virus by immunoglobulin A antibodies. Natl Acad. Sci. 89(15), 6901–6905 (1992).

43

Mestecky J, Russell MW, Elson CO. Intestinal IgA: novel views on its function in the defence of the largest mucosal surface. Gut 44(1), 2–5 (1999).

44

Doreau A, Belot A, Bastid J et al. Interleukin 17 acts in synergy with B cell–activating factor to influence B cell biology and the pathophysiology of systemic lupus erythematosus. Nat. Immunol. 10(7), 778–785 (2009).

45

Balu S, Reljic R, Lewis MJ et al. A novel human IgA monoclonal antibody protects against tuberculosis. J. Immunol. 186(5), 3113–3119 (2011).

46

Reljic R, Williams A, Ivanyi J. Mucosal immunotherapy of tuberculosis: is there a value in passive IgA? Tuberculosis 86(3), 179–190 (2006).

47

Ogra PL, Mucosal immunity: some historical perspective on host-pathogen interactions and implications for mucosal vaccines. Immunol. Cell Biol. 81, 23–33 (2003).

48

Nunn P, Williams B, Floyd K, Dye C, Elzinga G, Raviglione M. Tuberculosis control in the era of HIV. Nat. Rev. Immunol. 5(10), 819–826 (2005).

49

Wolf AJ, Desvignes L, Linas B et al. Initiation of the adaptive immune response to Mycobacterium tuberculosis depends on antigen production in the local lymph node, not the lungs. J. Exp. Med. 205(1), 105–115 (2008).

50

51

52

Reiley WW, Calayag MD, Wittmer ST et al. ESAT-6specific CD4 T cell responses to aerosol Mycobacterium tuberculosis infection are initiated in the mediastinal lymph nodes. Proc. Natl Acad. Sci. USA 105(31), 10961–10966 (2008). van Pinxteren LA, Cassidy JP, Smedegaard BH, Agger EM, Andersen P. Control of latent Mycobacterium tuberculosis infection is dependent on CD8 T cells. Eur. J. Immunol. 30(12), 3689–3698 (2000). Khader SA, Bell GK, Pearl JE et al. IL-23 and IL-17 in the establishment of protective pulmonary CD4 T cell responses


Review

after vaccination and during Mycobacterium tuberculosis challenge. Nat. Immunol. 8(4), 369–377 (2007). 53

Chackerian A, Alt J, Perera V, Behar SM. Activation of NKT cells protects mice from tuberculosis. Infect. Immun. 70(11), 6302–6309 (2002).

54

Schluger NW, Rom WN. The host immune response to tuberculosis. Am. J. Respir. Crit. Care Med. 157(3), 679–691 (1998).

55

Holmgren J, Czerkinsky C. Mucosal immunity and vaccines. Nat. Med. 11, S45–S53 (2005).

56

Mowat A, Faria A, Weiner H. Oral tolerance: physiologic basis and clinical applications. In: Mucosal Immunology. Ogra, PL, Mestecky J, Lamin ME, Strober W, Bienenstock JM, McGhee JR, Mayer L (Eds). Academic Press, CA, USA (1999).

57

Reyes V, Ye G, Ogra P, Garofalo R. Antigen presentation of mucosal pathogens: the players and the rules. Int. Arch. Allergy Immunol. 112(2), 103–114 (1997).

58

Ogra PL, Faden H, Welliver RC. Vaccination strategies for mucosal immune responses. Clin. Microbiol. Rev. 14(2), 430–445 (2001).

59

Bull CG, McKee C. Respiratory immunity in rabbits. VII. Resistance to intranasal infection in the absence of demonstrable antibodies. Am. J. Epidemiol. 9(2), 490–499 (1929).

60

Bessa J, Schmitz N, Hinton HJ, Schwarz K, Jegerlehner A, Bachmann MF. Efficient induction of mucosal and systemic immune responses by virus‐like particles administered intranasally: implications for vaccine design. Eur. J. Immunol. 38(1), 114–126 (2008).

61

Shim B, Choi YK, Yun C et al. Sublingual immunization with M2-based vaccine induces broad protective immunity against influenza. PLoS ONE 6(11), e27953 (2011).

62

Mora JR, Iwata M, Eksteen B et al. Generation of guthoming IgA-secreting B cells by intestinal dendritic cells. Science 314(5802), 1157–1160 (2006).

63

MacPherson G, Liu L. Dendritic cells and Langerhans cells in the uptake of mucosal antigens. Curr. Top. Microbiol. Immunol. 236, 33–53 (1999).

64

Macpherson A, McCoy K, Johansen F, Brandtzaeg P. The immune geography of IgA induction and function. Mucosal Immunol. 1(1), 11–22 (2008).

65

Mitragotri S. Immunization without needles. Nat. Rev. Immunol. 5(12), 905–916 (2005).

66

Ballester M, Nembrini C, Dhar N et al. Nanoparticle conjugation and pulmonary delivery enhance the protective efficacy of Ag85B and CpG against tuberculosis. Vaccine 29(40), 6959–6966 (2011).

67

Jensen K, Ranganathan UD, Van Rompay KK et al. A recombinant attenuated Mycobacterium tuberculosis vaccine strain is safe in immunosuppressed simian immunodeficiency virus-infected infant macaques. Clin. Vaccine Immunol. 19(8), 1170–1181 (2012).

68

Duerr A. Update on mucosal HIV vaccine vectors. Curr. Opin. HIV AIDS 5(5), 397–403 (2010).

69

Sack DA, Shimko J, Torres O et al. Randomised, doubleblind, safety and efficacy of a killed oral vaccine for


1011

Review Diogo & Reljic enterotoxigenic E. Coli diarrhoea of travellers to Guatemala and Mexico. Vaccine 25(22), 4392–4400 (2007). 70

Levine MM, Kotloff KL, Barry EM, Pasetti MF, Sztein MB. Clinical trials of Shigella vaccines: two steps forward and one step back on a long, hard road. Nat. Rev. Microbiol. 5(7), 540–553 (2007).

71

Davis S. Nasal vaccines. Adv. Drug Deliv. Rev. 51(1), 21–42 (2001).

72

Fine PE. BCG: the challenge continues. Scand. J. Infect. Dis. 33(4), 243–245 (2001).

73

Hesseling AC, Marais BJ, Gie RP et al. The risk of disseminated Bacille Calmette-Guerin (BCG) disease in HIV-infected children. Vaccine 25(1), 14–18 (2007).

74

Hesseling A, Johnson L, Jaspan H et al. Disseminated bacille Calmette-Guérin disease in HIV-infected South African infants. . Bull. World Health Organ. 87(7), 505–511 (2009).

75

76

White AD, Sibley L, Dennis MJ et al. Evaluation of the safety and immunogenicity of a candidate tuberculosis vaccine, MVA85A, delivered by aerosol to the lungs of macaques. Clin. Vaccine Immunol. 20(5), 663–672 (2013).

77

Wang J, Thorson L, Stokes RW et al. Single mucosal, but not parenteral, immunization with recombinant adenoviralbased vaccine provides potent protection from pulmonary tuberculosis. J. Immunol. 173(10), 6357–6365 (2004).

78

Hoft DF, Blazevic A, Stanley J et al. A recombinant adenovirus expressing immunodominant TB antigens can significantly enhance BCG-induced human immunity. Vaccine 30(12), 2098–2108 (2012).

79

80

Abel B, Tameris M, Mansoor N et al. The novel tuberculosis vaccine, AERAS-402, induces robust and polyfunctional CD4 + and CD8 + T cells in adults. Am. J. Respir. Crit. Care Med. 181(12), 1407–1417 (2010). Stylianou E, Diogo GR, Pepponi I et al. Mucosal delivery of antigen‐coated nanoparticles to lungs confers protective immunity against tuberculosis infection in mice. Eur. J. Immunol. 44(2), 440–449 (2013).

Santosuosso M, Zhang X, McCormick S, Wang J, Hitt M, Xing Z. Mechanisms of mucosal and parenteral tuberculosis vaccinations: adenoviral-based mucosal immunization preferentially elicits sustained accumulation of immune protective CD4 and CD8 T cells within the airway lumen. J. Immunol. 174(12), 7986–7994 (2005).

86

Xing Z, Lichty BD. Use of recombinant virus-vectored tuberculosis vaccines for respiratory mucosal immunization. Tuberculosis 86(3), 211–217 (2006).

87

Goonetilleke NP, McShane H, Hannan CM, Anderson RJ, Brookes RH, Hill AV. Enhanced immunogenicity and protective efficacy against Mycobacterium tuberculosis of bacille Calmette–Guerin vaccine using mucosal administration and boosting with a recombinant modified vaccinia virus Ankara. J. Immunol. 171(3), 1602–1609 (2003).

88

McShane H, Pathan AA, Sander CR et al. Recombinant modified vaccinia virus Ankara expressing antigen 85A boosts BCG-primed and naturally acquired antimycobacterial immunity in humans. Nat. Med. 10(11), 1240–1244 (2004).

89

Tameris MD, Hatherill M, Landry BS et al. Safety and efficacy of MVA85A, a new tuberculosis vaccine, in infants previously vaccinated with BCG: a randomised, placebocontrolled Phase 2b trial. Lancet 381(9871), 1021–1028 (2013).

90

Takahashi H, Sasaki K, Takahashi M et al. Mutant Escherichia coli enterotoxin as a mucosal adjuvant induces specific Th1 responses of CD4 and CD8 T cells to nasal killed-bacillus Calmette–Guerin in mice. Vaccine 24(17), 3591–3598 (2006).

91

Flynn JAL, Chan J. Immunology of tuberculosis. Annu. Rev. Immunol. 19(1), 93–129 (2001).

92

Ohara N, Nishiyama T, Ohara-Wada N, Matsumoto S, Matsuo T, Yamada T. Characterization of the transcriptional initiation regions of genes for the major secreted protein antigens 85C and MPB51 of Mycobacterium bovis BCG. Microb. Pathog. 23(5), 303–310 (1997).

93

Macadam A, Arnold C, Howlett J et al. Reversion of the attenuated and temperature-sensitive phenotypes of the Sabin type 3 strain of poliovirus in vaccinees. Virology 172(2), 408–414 (1989).

94

Todoroff J, Lemaire MM, Fillee C et al. Mucosal and systemic immune responses to Mycobacterium tuberculosis antigen 85A following its co-delivery with CpG, MPLA or LTB to the lungs in mice. PLoS ONE 8(5), e63344 (2013).

95

Olsen AW, van Pinxteren LAH, Okkels LM, Rasmussen PB, Andersen P. Protection of mice with a tuberculosis subunit vaccine based on a fusion protein of antigen 85b and esat-6. Infect. Immun. 69(5), 2773–2778 (2001).

81

Reljic R, Sibley L, Huang JM et al. Mucosal vaccination against tuberculosis using inert bioparticles. Infect. Immun. 81(11), 4071–4080 (2013).

82

Pepponi I, Diogo GR, Stylianou E et al. Plant‐derived recombinant immune complexes as self‐adjuvanting TB immunogens for mucosal boosting of BCG. Plant Biotechnol. J. 12(7), 840–850 (2014).

96

83

Doherty TM, Olsen AW, Van Pinxteren L, Andersen P. Oral vaccination with subunit vaccines protects animals against aerosol infection with Mycobacterium tuberculosis. Infect. Immun. 70(6), 3111–3121 (2002).

Holmgren J, Svennerholm A. Vaccines against mucosal infections. Curr. Opin. Immunol. 24(3), 343–353 (2012).

97

Haile M, Schröder U, Hamasur B et al. Immunization with heat-killed Mycobacterium bovis Bacille Calmette– Guerin (BCG) in Eurocine™ L3 adjuvant protects against tuberculosis. Vaccine 22(11), 1498–1508 (2004).

Betancourt AA, Delgado C, Estévez ZC et al. Phase I clinical trial in healthy adults of a nasal vaccine candidate containing recombinant hepatitis B surface and core antigens. Int. J. Infect. Dis. 11(5), 394–401 (2007).

98

Zhu Q, Talton J, Zhang G et al. Large intestine-targeted, nanoparticle-releasing oral vaccine to control genitorectal viral infection. Nat. Med. 18(8), 1291–1296 (2012).

84

1012

Watanabe K, Matsubara A, Kawano M et al. Recombinant Ag85B vaccine by taking advantage of characteristics of human parainfluenza type 2 virus vector showed Mycobacteria-specific immune responses by intranasal immunization. Vaccine 32(15), 1727–1735 (2014).

85




99

Eyles J, Bramwell V, Singh J, Williamson E, Alpar H. Stimulation of spleen cells in vitro by nanospheric particles containing antigen. J. Control. Release 86(1), 25–32 (2003).

100 Eyles J, Bramwell V, Williamson E, Alpar H. Microsphere

translocation and immunopotentiation in systemic tissues following intranasal administration. Vaccine 19(32), 4732–4742 (2001). 101 Carpenter ZK, Williamson ED, Eyles JE. Mucosal delivery

of microparticle encapsulated ESAT-6 induces robust cellmediated responses in the lung milieu. J. Control. Release 104(1), 67–77 (2005). 102 Mbow ML, De Gregorio E, Valiante NM, Rappuoli R. New

adjuvants for human vaccines. Curr. Opin. Immunol. 22(3), 411–416 (2010). 103 Holmgren J, Adamsson J, Anjuère F et al. Mucosal adjuvants

and anti-infection and anti-immunopathology vaccines based on cholera toxin, cholera toxin B subunit and CpG DNA. Immunol. Lett. 97(2), 181–188 (2005). 104 Giannini SL, Hanon E, Moris P et al. Enhanced humoral

and memory B cellular immunity using HPV16/18 L1


Review

VLP vaccine formulated with the MPL/aluminium salt combination (AS04) compared to aluminium salt only. Vaccine 24(33), 5937–5949 (2006). 105 Shimizu T, Sasaki K, Kato M et al. Induction of thymus-

derived gammadelta T cells by Escherichia coli enterotoxin b subunit in peritoneal cavities of mice. Clin. Diagn. Lab. Immunol. 12(1), 157–164 (2005). 106 Chong CSW, Cao M, Wong WW et al. Enhancement

of T helper type 1 immune responses against hepatitis B virus core antigen by PLGA nanoparticle vaccine delivery. J. Control. Release 102(1), 85–99 (2005). 107 Haile M, Hamasur B, Jaxmar T et al. Nasal boost with

adjuvanted heat-killed BCG or arabinomannan–protein conjugate improves primary BCG-induced protection in C57BL/6 mice. Tuberculosis 85(1), 107–114 (2005). 108 Yoon HA, Aleyas AG, George JA et al. Modulation of

immune responses induced by DNA vaccine expressing glycoprotein B of pseudorabies virus via coadministration of IFN-γ-associated cytokines. J. Interferon Cytokine Res. 26(10), 730–738 (2006).


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