On the other hand, low dose (red dots, B) and repeated exposure to natural allergens via mucosal surfaces leads to a Th2-driven allergic response. Although several methods of loading allergens into BGs are currently under various stages of development, the most popular approach involves first anchoring BG’s inner membranes with streptavidin.55 Following lyophilization, streptavidin-bearing BGs can then be loaded with allergens by re-suspending them in solution containing the desired biotinylated allergen of interest. living counterparts.22,26 Thus, save for the formation of trans-membrane channels, the morphology of resultant BGs including all cell surface structures remain unchanged as a result of the lysis events. Unlike convectional vaccines that need refrigeration, once produced and purified, BGs Tubastatin A HCl can be stored for several years at ambient room temperature as lyophilized products without loosing their potency.23 Open in a separate window Figure 1. Formation of Bacterial Ghosts Controlled expression of gene E leads to the lysis of bacteria and formation of a transmembrane tunnel spanning through the inner and outer membrane of bacteria. It is through the trans-membrane tunnel that the bacterial cell contents are expelled. Benefits of using BGs in allergen immunotherapy Since they share functional epitopes on their surfaces with their living counterparts, BGs can be used as excellent delivery systems of allergens.27 As previously demonstrated, heterologous proteins can be carried onto the BGs outer membranes (OM), the periplasmic space (PPS), inner-membrane (IM) or in the hollow lumen.28 Their ability to bear foreign peptides either on their surfaces or in the interior not only render them as targets for major histocompatibility complex class II antigen processing and presentation pathways by APCs,29 but also enables them to be combined with other display platforms such as Tubastatin A HCl the autotransporter systems.30 BGs intact pathogen associated molecular patterns (PAMPs) provide them with the original targeting functions from the host organism’s pattern recognition receptors (PRRs) as well as enhancing the activation of macrophages and DCs.31 Particulate substances Rabbit Polyclonal to EGFR (phospho-Tyr1172) are readily taken up by APCs since allergen uptake is dependent on several properties among them receptor interactions, surface charge, size, shape, hydrophobicity and hydrophilicity,32,58 Hence due to their inherent particulate nature, BGs tend to interact better with APCs compared with soluble allergens. Displaying allergens on bacterial ghosts membrane surfaces Allergen surface display entails the presentation of allergens or their hypoallergenic variants on the surfaces of BG shells. The first report documenting bacterial display of foreign peptides was published in 1986 when a 15 amino acid gene fragment was displayed in an accessible form onto an ((Der p) major allergens Der p 1 and Der p 2 in house dust mite allergic patients, it was found that immunotherapy using Der p allergens mixed with bacterial extracts was more effective at reducing allergy symptoms than Der p allergens administered alone.38 Building up from this phenomenon, Bohle et al. demonstrated that a recombinant fusion protein rSbsC-Bet v 1, consisting of Bet v 1 allergen and a bacterial surface protein SbsC exhibited a reduced histamine releasing capacity and a cytokine profile skewed toward a Th1 response.39 In addition, chemically linking Bet v allergens to bacterial proteins was also shown to display strong Th1-promoting capabilities from birch pollen-allergic individuals in vitro40,41 proving that bacteria or their components could be incorporated in immunotherapy.42 However, in most of these earlier studies, the allergens were produced as recombinant proteins intracellularly and recovery from whole cell extracts is a cumbersome multistep down streaming procedure. On the contrary, displaying of allergens on bacterial surfaces can be an easier and better strategy compatible with continuous culturing.43,44 Using recombinant DNA technology, allergens can be incorporated onto the bacterial envelope complexes before lysis and made to become elements of the BGs with heterologous proteins biophysical and functional properties being retained.45 Further, heterologous surface allergens that are exposed on the surface of bacteria elicit a superior immune response than those present intracellularly.46,47 From the earlier studies, it sounds logical to speculate that BGs may also be able to display allergen molecules or their hypoallergenic variants on their surfaces for allergen immunotherapy. Hence, it is possible to design BGs displaying therapeutic allergens for AIT Tubastatin A HCl with improved efficacy and safety in a similar manner described by Hjelm et al.48 Using this straightforward approach any allergen can theoretical be fused to proteins that have specific translocation and surface anchoring abilities like the autotransporter or ice nucleating systems. These systems can then be jointly expressed with lysis protein E in gram-negative bacteria such as E. coli. Thus, an allergen gene is fused to a specific translocation and surface anchoring protein gene (for example the hemoglobin protease in case of an autotransporter system) via the C-terminal in a plasmid downstream.
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