The protective role of Mtb infection against allergic asthma indicates the elucidation of the potential role of the TB vaccine in preventing asthma. The only licensed vaccine for TB is the BCG that is named after Albert Calmette and Camille Guérin who in 1908 attenuated the living virulent mycobacterium bovis (M. bovis) through continuous passages (231 passages) on culture media that lost the virulence and maintained the antigenicity of the organism. This vaccine was made throughout 13 years at the Institute Pasteur in Lille, France, and was used for the first time in humans in 1921 for TB prevention, with more than 100 million children vaccinated annually worldwide (27, 28). However, the protective efficacy of BCG is variable, ranging from 0% to 80% in different countries. Nonetheless, the exact reason for this variation remains unclear (27, 29, 30).

Interestingly, BCG is not a single strain, as the original strain in Pasteur institute generated several offspring strains via continuous passages in the countries that received the vaccine that reached 60 countries by 1927. For example, the Danish strain originated in 1931 in the name of 423rd transfer, whereas the Glaxo strain was derived from the 1077th transfer of the Danish strain. Many other strains were used, for instance; Pasteur, Tokyo, Prague, Russian, Moreau, …etc. (30, 31). The mechanism of the attenuation process of M. bovis resulting from the serial passages is not well interpreted. Nevertheless, it may involve deletion of a chromosomal region, called region of difference 1 (RD1), which is found only in virulent mycobacteria and absent in BCG, containing the encoding genes for ESAT-6 and CFP-10 and their secretion apparatus which are two fundamental virulence factors for M. bovis. Furthermore, other RD regions such as RD2, RD3 and RD14 to RD16 may be omitted. Moreover, single nucleotide polymorphisms are also involved in chromosomal changes (27, 28). Therefore, the genetic variability between the various BCG vaccine strains may explain the variable protection. Also, they induced different degrees of immune cell responses in vitro. However, there is no clear evidence that one strain produces more protection against pulmonary TB than the remaining strains (30). Another variable for the efficacy of BCG is the prior exposure to environmental mycobacteria, which are distributed differently among countries. These mycobacteria affect the efficacy of BCG through its pre-existing immune response that may block BCG replication, hence called the blocking hypothesis. In addition, it may induce a certain level of protection against TB, which masks any protective effect of subsequent BCG, thus called the masking hypothesis (28, 32).

BCG is considered very safe and the following reactions following intradermal (i.d.) administration are mild in the form of erythema and papule or ulceration that develops into scar, and generally do not require any treatment (33). BCG stimulates the immune response via binding its antigenic structures, such as lipoarabinomannan, phosphatidylinositol mannoside and trehalose 6,60-dimycolate, to toll-like receptor (TLR)2 and TLR4 on innate immune cells as macrophages that secrete IL-12 to induce Th1 cells polarization. The latter cells secrete IFN-γ that activates macrophages in a positive feedback loop (34, 35). Consequently, BCG is a potent inducer of Th1 cells which is, also, converted into memory cells to maintain life-long protection against Mtb infection. However, BCG, also, stimulates polarization of Tregs, as the response to Mtb infection, to prevent an exaggerated Th1 response (34). Due to the immunostimulatory effects of BCG, it is used as an immunotherapy for bladder cancer, multiple sclerosis and type 1 diabetes mellitus, and it also used as an adjuvant (35).

In experimental murine allergic asthma, we divided the potential effects of BCG into three categories ( Table 1 ): 1) preventive, 2) preventive and/or therapeutic, and 3) therapeutic, according to three time sets of vaccine administration: 1) before allergen sensitization (36–43), 2) with allergen sensitization (44–46), and 3) with/after aerosol allergen challenge (47, 48), respectively. In several studies, administration of BCG after birth or at an early age prevents the subsequent development of murine allergic asthma regardless of strain, route and number of doses (36–46). In addition, the BCG protective response is supported by the ability of BCG adoptive transferred stimulated dendritic cells (DCs) with different subsets to suppress the established allergic inflammation in murine asthma (37, 49–51). Moreover, the BCG administration after the establishment of allergic asthma still has the potential to suppress allergic inflammation and AHR (47, 48), indicating that BCG is not only a preventive agent, but also a therapeutic candidate for allergic asthma. Correspondingly, for human allergic asthma ( Table 2 ), several epidemiological studies with various study designs concluded that early BCG vaccination significantly decreases the subsequent development of asthma in different countries, such as England, France, Turkey, Germany and Spain in Europe, and Japan, Thailand and India in Asia (25, 52–57). Moreover, this conclusion was supported by the inverse correlation between BCG scar dimeter and atopic asthma in Brazil and Korea (65, 66). Interestingly, BCG scar diameter is a significant reflection of the immune response to BCG as a Th1 enhanced response with the subsequent increase of IFN-γ (66). Furthermore, BCG administration to asthmatic patients demonstrated therapeutic efficiency in the form of improved pulmonary functions and reduced medications. This occurs via the attenuation of Th2 response (67, 68), which proved the inhibitory effects of BCG even after developing atopic asthma.

The protective effect of BCG against allergic asthma can be mediated through two potential pathways ( Figure 1A ). First, promoted Th1 cells polarization with the subsequent increase in Th1/Th2 homeostasis along with their signature cytokines as IFN-γ/IL-5 ratio. This promotion could be induced by either polarization of naïve CD4+ to Th1 cells or switching of allergen specific Th2 into Th1 cells (38, 41–43). Another pathway is the upregulation of Tregs differentiation with the subsequent increase of IL-10, which is induced by interaction with stimulated TLR2, TLR4 and PD-1 that was expressed in DCs (37, 39, 40, 46). Interestingly, BCG can stimulate differentiation of naïve DCs into different subsets as CD8α⁺ and CD8α^- DCs that can induce both Tregs and Th1 cells through secretion of IL-10 and IL-12, respectively (37, 49–51). These two pathways provide protection in the form of suppression of Th2 cytokines secretion, eosinophilia, allergic inflammation, allergen specific-IgE and AHR (36–48). Moreover, BCG protected against asthma through reduced IL-17 production, the signature cytokine of Th17, that played a critical role in inducing neutrophilia and airway inflammation and correlated with AHR and disease severity (45, 69, 70). However, the complex action of Th17 in eosinophilic airway inflammation requires further elucidations.

On the contrary, several epidemiological studies found that early BCG vaccination did not decrease the risk of subsequent development of atopic asthma, these studies were performed in European countries such as Germany, Sweden, Netherland and a North American country; Canada ( Table 2 ) (58–64). However, there weren’t adequate animal research that supported this claim unlike the BCG protective effects that were reinforced by several animal studies as mentioned previously. The discrepancy between the preventive role of BCG vaccination in the development of human asthma reported in different countries is attributed to some factors. First, BCG strains contrasted with different immunogenicity (28); however, as was shown in Table 2 , the lack of information about the specific strains used in six of the 14 studies that supported (53–55) or did not support (59, 61, 62) the negative association between asthma and BCG, resulted in a severe difficulty to interpret the role of strains.

Nonetheless, four of seven studies ( Table 2 ) supported the protective role of BCG against asthma and reported strains, such as Pasteur (52), Tokyo 172 (25) and Copenhagen 1331 strains (56, 57), while the latter induced poor protective effects. Interestingly, for the seven studies that did not support the protective role of BCG ( Table 2 ), Copenhagen strain 1331 was utilized in three studies in Germany (60), the Netherlands (63) and Sweden (58). However, the other three studies, which did not mention the strain were also performed in the same three countries (59, 61, 62), with some potential to use the Copenhagen strain. The effect of this particular strain has not been investigated through animal research regarding its effect on murine allergic asthma unlike other strains shown in Table 1 . Thus, among the different BCG strains, Copenhagen strain may not induce protection against allergic asthma, and this requires further investigations. In addition, the seventh non-supportive study used the Pasteur strain and was conducted in Canada (64). However, the study did not exclude the subjects who received the vaccine after the neonatal period. Consequently, this leads to the second point of the controversy, which is that late administration of BCG during the neonatal period may increase the chances of prior exposure to environmental mycobacteria, that might decrease the efficacy of BCG as previously mentioned. In same context, another study was implemented in Sweden that did not exclude receivers (59).

Atopic hereditary can potentially interfere with the effects of BCG, particularly in the seven studies that did not support the protective function. Four of these seven studies did not detect a significant difference between vaccinated and non-vaccinated or asthmatic and non-asthmatic subjects regarding family history of atopy (58, 60, 63, 64). Three other studies did not apply this comparison, which weakens the potential interference of the genetic background with the BCG effects in those studies. In contrast, studies demonstrating the protective effects of BCG on allergic asthma in developed countries such as England and France (53, 54), which consequently contradicts the assumption that the preventive effects of BCG against asthma are mediated by the natural infection with Mtb in developing countries, where the incidence of tuberculosis is high, and not by the BCG itself (58). However, the preventive role of Mtb infection against the development of asthma, which was previously mentioned, is not controversial as BCG. Interestingly, Mtb infection generates four times more IFN-γ than BCG (45). Despite the widespread use of BCG as a TB vaccine, TB remains the leading cause of death from an infectious agent worldwide (71). Therefore, imitating natural Mtb infection through enhancing the efficacy of BCG and its administration at the neonatal period may induce protection against asthma and end this controversy.

In general, the conventional method to increase the efficacy of vaccines is the booster dosing (72), since repeated exposure to both antigen and polarizing cytokine is required for an effective immune response (73). In this regard, the BCG efficacy ranges between 0-80% and wanes over time (29), and it is classified as moderately effective vaccine (74). In addition, repeated intradermal injection of live attenuated vaccine is not acceptable due to the associated adverse effects. Thus, intranasal administration of recombinant immunodominant proteins is a better option (75). Antigen 85A (Ag85A) is a major immunodominant secretory protein in both Mtb and BCG (76), and can be incorporated with Mtb32 protein (an immunodominant mycobacterial protein) into an adenoviral vector, as its intranasal administration to asthmatic mice significantly suppresses allergic airway inflammation compared to BCG effect. This suppression occurs via increased IFN-γ and IL-10 and decreased IL-4, IL-5, IL-13 and IL-33 (77). In addition, antigen 85B (Ag85B), which is another major secretory mycobacterial protein, was recombined with pMG plasmid and administrated nasally to asthmatic mice inducing a protective effect by increasing IFN-γ/IL-4 ratio (75). Moreover, general Mtb secretory proteins, particularly Ag85A, have showed promising results for TB prevention in animals and humans and may replace the primary vaccine in the near future (76, 78, 79). It is expected that developed countries will replace the primary BCG vaccine with the recombinant protein vaccine because they cannot see any necessity for injecting live attenuated BCG concurrent with their low TB prevalence (60). However, there is a growing need for further investigation of intranasal use of Ag85A and Ag85B in humans. Additionally, intranasal administration of recombinant BCG producing genetically detoxified S1 subunit of pertussis toxin can prevent murine allergic asthma by increasing IL-12 and IFN-γ (80). Another method to increase BCG efficacy is the combination of BCG with CpG oligodeoxynucleotide, which is a synthetic TLR9 agonist that stimulates both macrophages (classical type) and DCs with subsequent induction of Th1 response. This combination inhibits allergic inflammation in murine asthma compared to BCG alone and may be a protective candidate effective for allergic asthma (34). In addition, it induces more protection against murine TB compared with BCG alone (81). Likewise, recombinant BCG strains producing IL-12 or IL-18 can further mediate protection against murine allergic asthma by switching from a Th2 response to a Th1 response (82, 83). All these genetic engineering-based modifications of BCG can improve its efficacy and provide enhanced protection against murine allergic asthma when administrated at early age. Thus, their use as boosters or as primary candidate vaccines for the prevention of human atopic asthma is promising for preventing one of the most prevalent chronic disease in the world and must be considered in future research.

It is unlikely that infection with live worms or their eggs could be delivered into children as a vaccine to prevent asthma (132). In order to solve this prospective issue, identification of helminth proteins that mediate anti-allergic properties, followed by the production of similar recombinant proteins that mediate the same immune responses observed in live worm infection would be a better option for developing a future vaccine for allergic asthma (143). Many commercial companies and private entities have produced and marketed helminth-derived molecules for the treatment of inflammatory diseases (144). Therefore, several studies demonstrated the role of different helminthic proteins in the prevention and therapy of murine allergic asthma ( Table 5 ). One of these effectual proteins is S. mansoni schistosomula (Smteg), extracted from the outer layer of the parasite. Smteg administration reduces eosinophilia, Th2 cytokines and allergen specific-IgE, and these effects can be mediated by a high level of IL-10 secreted by alveolar M2 macrophages and DCs. This indicates a potential preventive and/or therapeutic effects of Smteg protein in asthmatic mice (146). In addition, Sm22·6, which is a soluble protein associated with the tegument of S. mansoni, induces the same suppressive effects as Smteg through the induction of IL-10 secreted by Tregs. PIII, also derived from S. mansoni, mediates the same effects, but without elevation of IL-10. In addition, S. mansoni-derived Sm29 induces suppressive effects; however, lesser than the other two molecules (145). Another schistosome is S. japonicum containing the SJMHE1 protein, which is an HSP60‐derived peptide. SJMHE1 administration suppresses the development of murine asthma through inhibiting eosinophilia and Th2 cells and inducing Th1 and Treg cells (149). Likewise, soluble S. japonicum egg antigen mediates the same effects without inducing Th1 response (147). While P6, P25, and P30 peptides in SjP40 protein, which is the dominant protein of S. japonicum eggs, could prevent murine asthma by activating Th1 and alleviating Th2 cells (148). For A. lumbricoides, total protein extracts reduce IL-5 and eosinophilia and induce IL-10 to protect mice against experimental asthma (150). Another type of ascaris is A. suum that contains three different components. Each component can prevent murine asthma, namely; total protein extracts (151), pseudocoelomic fluid (152) and PAS-1 (142). They reduce eosinophilic inflammation and Th2 activities through the induction of Tregs and Th1 (142) or inhibition of DCs (152). Interestingly, as mentioned previously, A. suum has allergenic protein that can induce and exacerbate allergic reaction (142), indicating that identifying effective anti-allergic components in the worm extracts would be a crucial approach for developing novel preventive strategies. Moreover, soluble extracts of adult worms of T. spiralis have more potent suppressive impact on allergic asthma than those of soluble extracts of the larvae muscle of the same helminth (11). Another important consideration is the timing of protein injection, as the effective suppressive action of some helminth derived protein depends on early administration prior to developing asthma, which also indicates the importance of elucidating the preventive and therapeutic potentials of each candidate protein. The excretory/secretory products of Fasciola hepatica are a clear example of this indication, the administration of which during experimental murine asthma development, induces significant suppressive effects, while their administration after the establishment of the disease has no apparent effect (165). Another significant protein derived from helminth is AIP-2 secreted by Ancylostoma caninum (hookworm). AIP-2 has both preventive and therapeutic potentials on murine asthma through inhibition of DCs and induction of Tregs (164). ES-62, another promising molecule secreted by the filarial nematode Acanthocheilonema viteae (A. viteae). ES-62 has preventive and/or therapeutic potentials through the direct inhibition by mast cells FcϵRI-induced release of allergy mediators through blocking of key signal transduction molecules (153, 155). Moreover, several proteins derived from different species of helminths demonstrate preventive or therapeutic abilities against murine allergic asthma, such as Trichuris suis (162), H. polygyrus (156, 157), N. brasiliensis (158), Caenorhabditis elegans (161), Clonorchis sinensis (160), Angiostrongylus cantonensis (163) and Toxascaris leonine (159) ( Table 5 ). Thus, worm-derived proteins might be exploited to prevent allergic asthma, since they suppress asthma through the same mechanisms that are induced with living worm infections without entailing their undesired side effects.

There are many studies investigating the protective effects of helminth-derived proteins in murine allergic asthma. Nevertheless, there are only few studies that detected these protein actions in human allergic asthma. S. mansoni antigens, Sm29 and Sm29TSP-2 reduce Th2 cells while inducing Tregs with subsequent high IL-10 production in the cell cultures from asthmatic patients (166). In same vein, other S. mansoni recombinant antigens as Sm22.6, Sm14, P24, and PIII antigen increase IL-10 secretion in cell cultures from subjects with asthma (167). Additionally, AIP-2, from the hookworm, suppresses human DCs activation and proliferation from asthmatic subjects in-vitro (164). In addition, ES-62 from A. viteae when co-cultured with sensitized human mast cells from healthy subjects, mediates the same effects previously mentioned with ES-62 with murine mast cells (153). However, these studies only demonstrate in-vitro therapeutic potentials for human asthma. Therefore, further research is needed to investigate both in-vitro and in-vivo preventive potentials of helminth-derived proteins against the development of human allergic asthma, to design novel strategies for potential allergic asthma vaccines.