Review

Bacillus subtilis comes of age as a vaccine production host and delivery vehicle Expert Review of Vaccines Downloaded from informahealthcare.com by Nyu Medical Center on 06/04/15 For personal use only.

Expert Rev. Vaccines Early online, 1–14 (2015)

Sergio Rosales-Mendoza* and Carlos Angulo* 1 Laboratorio de biofarmaceuticos recombinantes, Facultad de Ciencias Quı´micas, Universidad Autonoma de San Luis Potosı´, Av. Dr. Manuel Nava 6, SLP, C.P. 78210, Mexico 2 Centro de Investigaciones Biologicas del Noroeste, SC. Instituto Politecnico Nacional 195, Playa Palo de Santa Rita Sur, La Paz, B.C.S. C.P. 23096, Mexico *Authors for correspondence: Tel.: +444 826 2440 [email protected]; [email protected]

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Bacillus subtilis is a vaccine production host and delivery vector with several advantages such as a low production cost, straightforward administration as it is safe for human consumption and the production of spores exerting adjuvant effects. This review summarizes the expression approaches and provides an updated outlook of how a myriad of pathogens have been targeted under this technology. Furthermore, by reviewing the literature, several promising candidates in terms of immunogenic and immunoprotective potential have been identified. The immune profiles achieved comprise either humoral or cellular responses, which reflect versatility for application in the fight of distinct pathologies that demand specific polarization on the immune responses. Some perspectives for this field are also envisioned. KEYWORDS: cytotoxic lymphocyte . delivery vehicle . intranasal immunization . low cost vaccine . mucosal vaccines .

oral immunization

.

particulate antigen

.

spore

.

sublingual immunization

Vaccinology faces important challenges since the efficacy of vaccines against several pathogens of relevant epidemiology impact has not been addressed. That is the case of HIV/AIDS for which only a candidate vaccine has shown marginal protection in clinical trials [1]. In addition, the BCG vaccine fails to protect adults against active forms of tuberculosis (TB) resulting in millions of deaths each year [2]. In contrast, this vaccine in infants has prevented TB (0–80% efficacy). In addition, several vaccine-preventable diseases still cause considerable burden [3,4]. On the other hand, chronic non-infectious diseases are the predominant challenge to global health accounting for nearly two-thirds of deaths worldwide [5]. Therefore, platforms offering low costs and proper immune profiles and immunoprotection are highly attractive and must be pursued in the development of new vaccines, especially to support health programs in developing countries. One important trend in vaccinology comprises the development of needle-free vaccines, which offer a myriad of advantages such as: aid in mass vaccinations by increasing ease and speed of delivery, provide improved safety and compliance, decrease costs and reduce pain associated with vaccinations. In addition, needlefree vaccines can induce immune responses at

10.1586/14760584.2015.1051469

both systemic and mucosa levels [6,7]. When intranasal immunization is performed, potent immune responses are observed at mucosal and systemic levels; however, one disadvantage is the possible migration of the antigen to the CNS enhancing the risk of adverse side effects. Oral vaccination is perhaps the most attractive approach in terms of easy administration and protection against enteric pathogens, since systemic and local immunity can be achieved; however, oral vaccine development is often hindered by unfavorable physicochemical properties of protein antigens causing degradation in the gastrointestinal tract and poor transport across the intestinal wall. Another challenge involves overcoming tolerance that is frequently induced under oral immunization schemes [8]. Developing new approaches to overcome these obstacles will provide new vaccines and delivery systems capable of eliciting strong protective immune responses [9,10]. In this arena, an attractive alternative consists in developing vaccine production platforms based on expression hosts that fall in the Generally Recognized As Safe (GRAS) status, which can serve as low-cost biofactories and at the same time as oral delivery vehicles without the need for complex purification steps, thus allowing for a straightforward

 2015 Informa UK Ltd

ISSN 1476-0584

1

Review

Rosales-Mendoza & Angulo

low-cost oral vaccine formulation. Under this scheme, the GRAS organisms explored thus far are plant cells, algae, yeast and bacterial species such as Lactobacillus and Bacillus subtilis [11–16]. In particular, this review provides an updated outlook on the B. subtilis-based vaccines development and identifies the potential and the perspectives for this field.

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Why using B. subtilis for vaccine production & delivery?

Several advantages are identified in B. subtilis both as an expression host and as a delivery vehicle of subunit vaccines: spores constitute a proven vaccine delivery vehicle for oral, sublingual and nasal administration with immunogenic responses [15]; spores exert adjuvant activity which is of special importance given the poor immunogenicity shown by most of the orally administered antigens [17]; spores are able to germinate in the mice gut, a fact that has been associated with the capability to induce strong mucosal immune responses allowing for the intracellular presentation of antigens when they germinate inside antigen-presenting cells (APCs); being killed in a short period of time [18–20], although the induction of cellular immune responses can be also achieved in the absence of spore germination in the gut [21]; spores can be produced at low cost when compared with conventional systems based on complex downstream processing that require removal of endotoxins from the host, for example, in the case of lipopolysaccharides from Escherichia coli; spores are stable at ambient temperature and thus do not require refrigerated storage and transport, which also dramatically reduces vaccine cost; B. subtilis is safe for human consumption and has been categorized as GRAS by the FDA, currently being used as probiotic and the basis for food additives which implies that production platforms are already implemented with the potential to accelerate scaling-up during vaccine production [22]; B. subtilis has an important set of genetic engineering tools available, facilitating the development of vaccine prototypes [23]. Among the conventional platforms for developing vaccines, viral vectors are convenient because they are in general highly immunogenic [24]. However, the following disadvantages are identified for these systems: high production cost, risk of pathogenesis in specific individuals, pre-existing immunity, generation of replication-competent virus and potential for tumorigenesis [25]. On the other hand, although E. coli-based vaccine platforms are characterized by high yields, these require extensive and costly purification processes to eliminate endotoxins [26]. In contrast, B. subtilis overrides these limitations since it constitutes a safe organism with no possibilities to become pathogenic or containing toxic compounds. Therefore, B. subtilis offers the potential to serve in the development of new attractive vaccines in terms of safety, needle-free administration, thermostability and costs. So far, several groups have assessed the expression of a myriad of antigens with promising evidence in terms of immunogenicity and immunoprotection in preclinical trials, most of them administered by mucosal routes. doi: 10.1586/14760584.2015.1051469

B. subtilis vaccine design & antigen expression modalities

Antigen displayed on vegetative cells: This is a typical approach to address antigen presentation using bacterial species which is accomplished by displaying the target antigen through genetic fusion with surface proteins. In the case of Gram-positive bacteria, the S-layer proteins containing LPXTG motifs or PgsA are typical targets for this purpose [27]. However, this method has the limitation that some antigens may not be suitable for fusion and secretion. B. subtilis strains for this modality are typically generated using constitutive promoters such as the PrrnO promoter, which drives expression in vegetative cells. The spores derived from this type of strains can be used for immunization and can germinate inside host phagocytic cells allowing an efficient antigen presentation process. Intracellular located antigen in vegetative cells: B. subtilis strains express and accumulate the antigen into the cytosol. Antigens displayed on spores. This is the most explored alternative for vaccination, which is typically achieved by fusing at the translational level the antigen sequence to an endogenous host spore surface protein [28]. Displaying antigens on the surface of a spore offers the advantage that this process does not require secretion through the membrane. This method has the unique requirement that the expression should occur during the sporulation phase of the mother cells, which is achieved by the use of sporulation-specific promoters such as those of the coat proteins. During this expression phase, chaperones facilitate proper folding of the chimeric protein. The sporulation comprises the formation of a polar septum, the engulfment of the forespore, which becomes a double membrane-bound structure; the cortex, which is composed of peptidoglycan [29], assembled between the inner and outer forespore membranes and the proteinaceous coat, which is the outermost spore layer [30,31]. The central part of the spore is the core, containing partially dehydrated cytoplasm including the chromosome (FIGURE 1) [32,33]. B. subtilis spores contain at least 70 individual proteins with five proteins crucial for proper spore coat formation (SpoIVA, SpoVM, SpoVID, SafA and CotE) called, therefore, morphogenetic proteins [34]. Other three proteins called CotX, CotY and CotZ have shown to be morphogenes of the crust [35]. The spore coat proteins typically used for displaying antigens on B. subtilis spores include CotB [28], CotC [36], CotG [37] and CotX (FIGURE 1) [38]. Interestingly, the display of two heterologous proteins in a single spore has been achieved [39]. Spores can survive transit across the gastrointestinal tract, germinate and undergo limited rounds of replication and cell growth in the intestine before being excreted. Antigens adsorbed on the spore surface: This recent method relies in the absorptive capacity of the spore surface thanks to physicochemical interactions. Therefore, a pure antigen is adsorbed on spores from a wild-type B. subtilis strain and used for immunization [40]. A modification of this method was further developed consisting of the assembly and display of the target antigen synthesized in the mother cell compartment but outside the spore as it occurs for the endogenous coat proteins [41]. This method comprises Expert Rev. Vaccines

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Bacillus subtilis comes of age as a vaccine production host and delivery vehicle

Review

the expression of the heterologous protein Crust Outer coat in the mother cell compartment during Inner coat the late sporulation phase through the Basement layer sporulation-specific cry1Aa promoter. Outer forespore membrane A subsequent attachment or adsorption Cortex onto the spore surface, which acts as a Inner forespore membrane non-selective adsorbing matrix, is viable Core Chromosome thanks to the hydrophobic and highly electrostatic nature of the spore surface. This concept was successfully implemented for Genetically-fused target CotB carboxymethylcellulase and b-galactosidase antigen CotC as models of monomeric and multimeric Endogenous anchor-coat protein CotG A enzymes, respectively, and could be CotX applied in theory to vaccine antigens [42]. Antigen adsorbed, expressed in the late The adsorption methodologies have been sporulation phase improved by the use of mutants for the B cotH gene, having a strongly altered spore coat with higher absorption capacity than Figure 1. Graphical representation of expression approaches for antigens in the wild-type strains [43]. Bacillus subtilis spores. Spore structure comprises multiple layers of the spore that surIn addition to antigen expression round the genome located at the central core. a. Antigens can be displayed on the crust approaches, one important trend in this through genetic fusion with coat proteins. b. During the late sporulation phase, antigens can be assembled and displayed on the mother cell compartment but outside the spore, field comprises the development of being adsorbed on the spore surface thanks to its hydrophobic and highly electrostatic improved strains for vaccine development nature. purposes. For example, a novel approach has been recently published regarding the use of strains genetically engineered to provide improved gut ability to adhere onto the intestinal epithelium and accumulate adhesion through the expression of the Yersinia pseudotuberculo- in the gastrointestinal tract of shrimp [49] as observed in human sis InvA, which interacts with the b-integrin receptor promot- beings and mice. An important observation is the capacity of ing the invasion of gut epithelial cells [44], and the Lactobacillus spore germination to produce a vegetative cell in the lumen of brevis S-layer protein that mediates binding to fibronectin [45]. the gastrointestinal tract or in the gut-associated lymphoid tisThese proteins were expressed as fusions with the N-terminal sue, which has been proven by the fact that a significant numportion of the CotB protein. Notably, this approach enhanced ber of spores, as well as germinated spores, have been recovered serum and salivary antibody responses to the target Streptococcus in the Peyer’s patches and mesenteric lymph nodes in sporemutans antigen P139-512 and thus constitute a new tool that immunized animals [18]. An important goal for vaccine development comprises finding adjuvants capable of inducing strong will open new avenues for vaccine development. Th1-polarized responses, which are required to elicit protective immunity in many infectious diseases. In this regard, B. subtilis Adjuvant activity of B. subtilis spores Most protein antigens are poorly immunogenic when delivered spores can induce Th1 immune responses characterized by eleby the oral route because of degradation by gastric secretions, vated IFN-g-producing T cells, which are similar in potency to therefore strong mucosal adjuvants in oral vaccine formulations monophosphoryl lipid A, an adjuvant extensively used to test are needed. Thus, a delivery vehicle with additional mucosal TB vaccines in both preclinical and clinical trials [50]. In addiimmunogenic properties is highly recommended. Currently, tion, T and B cells, natural killer cells, macrophages, hemocytes there is a concerted effort to develop adjuvants that avoid side (shrimp) and dendritic cells are stimulated by B. subtilis. The effects such as toxicity. B. subtilis spores and vegetative cells are possible immune molecular mechanisms of B. subtilis recogniable to stimulate proliferation of immune cells within the gut- tion, processing and effector responses are currently unknown. associated lymphoid tissue and promote potent immune A possible mechanism that has been described for Bacillus responses [18,46]. The ability of spores to germinate in this anthracis spores comprises the Toll-like receptor 2 activation important region makes them attractive for reinforcing vaccine- via MyD88, resulting in the activation of the NF-kB pathway induced immunity. It has been demonstrated that following the with the subsequent cytokine expression [51]. What have been uptake of spores by M cells, they can interact with lymphocytes documented for B. subtilis are the systemic IgG isotypes and and APCs in the Peyer’s patches with the subsequent induction mucosal secretory immunoglobulin A (sIgA) responses, as well of humoral and cellular responses in vertebrates (FIGURE 2) [47]. as the secretion of IL-12, IL-2, IL-6, IL-10, IFN-g and TNF-a Moreover in invertebrates, which do not possess adaptive cytokines that can be strongly induced and even provide proimmune system, B. subtilis spores have been able to stimulate tection against specific pathogen infection [52]. These immune phagocytosis and immunoprotection [48], probably due to their responses depend on the B. subtilis strain, host, scheme and informahealthcare.com

doi: 10.1586/14760584.2015.1051469

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Rosales-Mendoza & Angulo

Vaccine design approaches A Spores expressing target antigen fused to coat proteins, generated by the use of promoters activated during sporulation

B

C

Spores from strains that express Spores from WT strains in the antigen in the cytosol of which pure antigen has been vegetative cells under the control absorbed on the spore surface of a constitutive promoter

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Mucosal administration

Spores capable to colonize gut through the expression of adhesines or mutants with enhanced mucoadhesive properties can improve vaccine efficacy

M cell

DC

B-cell

Peyer’s patch

lgA Plasma cell

T-cell

Under strategy B, spores may germinate and express antigen into dendritic cells, leading to intracellular presence of the antigen, which favors the induction of CTLs responses

CTL

Blood circulation B-cell

lgG

Mesenteric lymph node Plasma cell T-cell CTL

Figure 2. Schematic representation of the strategies and mechanisms implicated on the Bacillus subtilis-based vaccines development. Distinct vaccine designs are represented (A, B and C panels) and innovative approaches, such as the recombinant expression of proteins with gut adhesive properties, to improve vaccine efficacy are indicated. B. subtilis has a proven capacity to induce cellular and humoral immune responses at both systemic and mucosal levels. CTL: Cytotoxic T lymphocyte; DC: Dendritic cell; WT: Wild-type.

mucosal route of immunization. Therefore, the broad spectra of immune responses elicited in combination with the associated benefits of safety suggest that B. subtilis could be appropriate for improving the immunogenicity of various vaccines [47]. Several results encourage the use of B. subtilis as a natural adjuvant for vaccine formulations for mice, rats, swine, shrimp, crayfish and human beings. Overall, B. subtilis spores could represent an excellent vaccine adjuvant because they are relatively straightforward to produce, generally considered safe for humans and can be transported at ambient temperatures, which contribute to cost reduction [15]. Expert commentary TABLE 1 presents a compilation of the B. subtilis-based vaccines reported thus far, which reflects interesting findings. An analysis of the efforts shows that B. subtilis spores or vegetative cells can be used as a straightforward source of vaccines able to induce strong immune responses through the intranasal,

doi: 10.1586/14760584.2015.1051469

sublingual and oral routes. Remarkably, the majority of the B. subtilis-based vaccines have been evaluated in immunoprotection assays with promising findings, including prototypes against the following pathogens: Mycobacterium tuberculosis [53], Helicobacter pylori [54,55], White spot syndrome virus [48,49,56,57], Clonorchis sinensis [58,59], Clostridium tetani [60–63], Clostridium difficile [64], Foot-and-mouth disease virus [65], Rotavirus [66], Clostridium perfringens [67], B. anthracis [68] and enterotoxigenic E. coli [69]. Most of these vaccine prototypes listed above have targeted human pathogens, while few of them report animal pathogens. The latter comprise four candidates against White spot syndrome virus [48,49,56,57] and one candidate against the Foot-and- mouth disease virus [65]. In human vaccine candidates, preclinical evaluations have resulted in very positive outcomes, therefore, performing clinical trials will be the following step. While in the case of veterinary vaccines, open-field trials in the primary hosts are expected to be conducted in the following years. Expert Rev. Vaccines

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Surface expression by spore-coat C-terminus of CotB protein fused to MPT64 under control of CotB promoter

MPT64 antigen

Mice were immunized by the intranasal route in distinct immunization schemes: Live spores – One group received HU58 spores on days 15, 36 and 57 – One group was immunized by the intranasal route with spores on days 21, 42 and 63 – One group was primed with BCG vaccine on day 1 and boosted by the intranasal route with spores on days 42 and 63 Both approaches induced Th1 immune responses estimated in the IFN-g ELISPOT and polyfunctional T-cell analysis Prime-boost approach using either live or inactivated spores achieved protection in terms of bacterial load in the lungs Inactivated spores alone also showed immunoprotection in terms of bacterial load

Mice were immunized by the oral route for 3 consecutive days weekly on weeks 1, 3, 5. Fecal IgA and serum IgG responses were induced. Mice were orally challenged (3  108 CFUs of H. pylori) and a reduction of 84% in the stomach bacterial load was achieved

[53]

[55]

Several pathogens have been targeted through B. subtilis-based formulations leading to relevant outcomes in preclinical evaluations. BCG: Bacille Calmette–Guerin; CFA: Colonization factor antigen; CFU: Colony forming unit; CsLAP2: C. sinensis Leucine aminopeptidase; CT: Cholera toxin; ETEC: Enterotoxigenic E. coli; GST: Glutathione-S-transferase; IFN: Interferon; MPT: Mycobacterium tuberculosis; TTFC: Tetanus toxin fragment C; UreB: Urease B; WSSV: White spot syndrome virus.

Mycobacterium tuberculosis

Surface expression by spore-coat protein CotC fused to UreB under control of CotC promoter

UreB

H. pylori

[54]

Mice were immunized by the oral route on days 1, 3, 5, 22, 24, 26, 43, 45, 47. Strong immune cellular responses were induced when both types of spores were co-administered with spores displaying human IL-2

Intracellular expression in vegetative cells of the full-length UreB under the control of the rnnOP promoter Surface expression by spore-coat protein CotC fused to a fragment of UreB, or spore-coat protein CotB fused to human IL-2, under the control of CotC and CotB promoters, respectively

Full-length UreB (vegetative expression) Fragment of UreB (spore expression)

Helicobacter pylori

[44]

Mice were immunized with spores capable of binding to the gut epithelium by means of expressing bacterial adhesins. Oral immunization was performed on days 1, 2, 3, 15, 16, 17, 29, 30 and 31. Sublingual immunization was performed on days 1, 15 and 30. The adhesive spores increased the systemic and secreted antibody responses to the P1 protein in all mice groups, used as a model antigen, following oral, intranasal and sublingual administration. P1-specific antibodies efficiently inhibited the adhesion of the S. mutans to abiotic surfaces

B. subtilis strain was engineered to express the full-length Yersinia pseudotuberculosis InvA, which interacts with the b-1 integrin receptor and promotes the invasion of gut epithelial cells, and the Lactobacillus brevis S-layer protein (SlpA), involved in the binding to fibronectin and conferring the capability to colonize the mammalian gut, which were surface displayed as fusions with the Nterminal portion of the CotB protein under the control of the endogenous promoter The vaccine consisted of a strain that perform intracellular expression in vegetative cells of the P139-512 antigen from S. mutans under the control of PgsiB, a promoter active only during the vegetative growth stage

Truncated P1 protein (aa 139-512)

Streptococcus mutans

Ref.

Immunogenic properties

Expression approach

Antigen description

Target pathogen

Table 1. Summary of Bacillus subtilis-based vaccines.

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Bacillus subtilis comes of age as a vaccine production host and delivery vehicle

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doi: 10.1586/14760584.2015.1051469

doi: 10.1586/14760584.2015.1051469

Vp26 and Vp28 antigens

VP28 antigen

VP28 antigen

VP28 antigen

Enolase

22.3 kDategumental protein (CsTP22.3)

White spot syndrome virus

White spot syndrome virus

White spot syndrome virus

White spot syndrome virus

Clonorchis sinensis

C. sinensis

[56]

[57]

[58]

Shrimp (L. vannamei) were immunized by the oral route with spore-coated feed pellets every day over a 7-day period. A challenge was applied either 7 or 21 days after spore treatment. Relative survival rates for CotB-Vp28 and CotC-Vp28 was 37.9 and 44.8% (immediately), 46.4 and 50% (7 days postchallenge) and 30 and 33% (21 days post-challenge), respectively Crayfish were immunized through feeding coated food pellets for 20 days. Significant protection against WSSV was observed in groups treated with spores displaying VP28 and challenged 3 or 14 days post-vaccination (values of 65.4 and 73.9%, respectively). Protection was not significant in the vegetative cells expressing VP28 Sprague-Dawley rats were immunized by the oral route (5  1010 spores/rat) three-times on day 1, 14 and 28. Serum IgG was induced corresponding to a mixedTh1/Th2 response. Rats were challenged with C. sinensis metacercariae and a protective efficacy of 61.07% in worm reduction rate and 80.67% in egg reduction rate were achieved in the spore Enolase-treated group Rats were immunized by the oral route with spores on days 0, 1, 2, 16, 17, 18, 33, 34 and 35. Specific IgA responses were induced. Rats treated with spores displaying CsTP22.3 showed a reduction on worm counts (44.7%) and eggs per gram stool (30.4%) after challenge

Surface expression by spore-coat protein CotC fused to Enolase under control of CotC promoter of B. subtilis

Surface expression by spore-coat of CotC protein fused to CsTP22.3, expressed by the endogenous CotC promoter

Expression was based on secretion mediated by the secretion signal MMARKIAGMATSLLVIFSSSAVA and the strong s43 promoter for the expression of VP28. Spores (rVP28-bs) or vegetative cells (rVP28-bv) were obtained

Surface expression by spore-coat protein CotB or CotC fused to Vp28 under control of CotB or CotC promoter, respectively

[59]

[49]

Surface expression by spore-coat truncated protein CotB fused to VP28 under control of CotB promoter

Surface expression by spore-coat protein CotC fused to Vp26 or Vp28 under control of CotC promoter of B. subtilis

Shrimp L. vannamei were immunized by the oral route with 1  109 CFU/g pellet (fed doses comprised 0.1–0.2% of shrimp body weight) during 7, 14 or 21 days. Shrimps were challenged with WSSV and positive innate immune responses of muscle superoxide dismutase and heart prophenoloxidase activities as well as 47% of relative survival compared with untreated control were achieved

Ref. [48]

Immunogenic properties Shrimp (Litopenaeus vannamei) were immunized by the oral route (2  106 spores/g of pellets) for 1 week period (Exp. 1) or 2-month period (Exp. 2) three-times daily with a rate diet per weight. Shrimp were challenged with 1 g of WSSV-infected shrimp tissues. In Exp. 1, 100% of WSSV infected shrimp died, but a 5 days delay on cumulative mortality was observed in the spore-treated group. In Exp. 2, a 10% cumulative mortality was observed in the Vp28 spore-treated group, while 0% of mortality was observed in the Vp26 spore group (100% protection)

Expression approach

Several pathogens have been targeted through B. subtilis-based formulations leading to relevant outcomes in preclinical evaluations. BCG: Bacille Calmette–Guerin; CFA: Colonization factor antigen; CFU: Colony forming unit; CsLAP2: C. sinensis Leucine aminopeptidase; CT: Cholera toxin; ETEC: Enterotoxigenic E. coli; GST: Glutathione-S-transferase; IFN: Interferon; MPT: Mycobacterium tuberculosis; TTFC: Tetanus toxin fragment C; UreB: Urease B; WSSV: White spot syndrome virus.

Antigen description

Target pathogen

Table 1. Summary of Bacillus subtilis-based vaccines (cont.).

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Expert Rev. Vaccines

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Leucine aminopeptidase 2 (CsLAP2)

FliD protein

Carboxyterminal repeat domains of toxins A and B

L1 major capsid protein (L1)

TTFC

C. sinensis

Clostridium difficile

C. difficile

Human papilloma virus type 33

Clostridium tetani

[62]

[60]

Mice were immunized with vegetative cells by intranasal (i.n.) or sublingual (s.l.) routes, with or without addition of mutant heat labile toxin from enterotoxigenic Escherichia coli (mLT) as adjuvant. Immunizations were given three- to four-times at 2-week intervals. Mice were challenged with tetanus toxin 2 weeks after the last immunization. Both systemic and mucosal humoral responses were induced. s.l. immunization using mLT as adjuvant induced the strongest response. i.n. or s.l. immunized mice were challenged with tetanus toxin and 100% protection was achieved Piglets were immunized with (i) vegetative cells by the i.n. or s.l. routes, with or without mTL adjuvant; or (ii) spores by the oral or i.n. route. Scheme comprised 4 doses administered at 2-week intervals. s.l. or i.n. immunization induced neutralizing serum IgG responses and salivary and vaginal IgA responses. Oral administration was ineffective. A balanced Th1/Th2 response was observed in s.l. or i.n. immunized animals. 100% of protective efficacy in mice was observed when challenged with tetanus toxin pre-incubated with sera from piglets

Intracellular expression of TTFC under the control of Pspac1/2 constitutive promoter in B. subtilis vegetative cells

Intracellular expression of TTFC under the control of the Pspac1/2 constitutive promoter in B. subtilis vegetative cells or spores

[70]

[64]

Mice were immunized by the oral route with spores on days 0, 14, 35 and 57. Serum (IgG) and fecal (IgA) antibody production was induced. Hamsters were dosed. Hamster were dosed o.g. (0.2 ml) on days 0, 14, 35 and 57 with a dose of 1  1010 spores. Hamsters were o.g. infected with 100 C. difficile spores. A26-39 domain of toxin A conferred 100% of protection

Surface expression by spore-coat of CotB and CotC proteins fused to the toxin domains A26-39 and B15-24 under control of endogenous promoters (PcotB or PcotC, respectively)

Mice were immunized by the subcutaneous route with spores along Quil-A as adjuvant on weeks 0, 2 and 6. Seric IgG responses and IFN-g-producing spleen cells were induced

[72]

No oral immunization studies were performed. Spores were only characterized in terms of reactivity with an anti-FliD serum

Surface expression by spore-coat proteins CotB, CotC, CodG and CodZ, fused to FliD protein under the control of corresponding Cot promoters

Intracellular expression of L1 under the control of xyloseinducible promoter

[88]

Mice were immunized by the oral route during 3 consecutive days weekly on weeks 1, 3, 5. IgG/IgG1/IgG2a responses were induced in serum; IgA responses were induced in serum, intestinal lavage fluids and bile; Th1 and Th17 cellular immunity is proposed as effector mechanisms as evidenced by cytokine profiles observed in supernatants of splenocytes from immunized animals

Surface expression by spore-coat protein CotC fused to CsLAP2 under control of CotC promoter

Ref.

Immunogenic properties

Expression approach

Several pathogens have been targeted through B. subtilis-based formulations leading to relevant outcomes in preclinical evaluations. BCG: Bacille Calmette–Guerin; CFA: Colonization factor antigen; CFU: Colony forming unit; CsLAP2: C. sinensis Leucine aminopeptidase; CT: Cholera toxin; ETEC: Enterotoxigenic E. coli; GST: Glutathione-S-transferase; IFN: Interferon; MPT: Mycobacterium tuberculosis; TTFC: Tetanus toxin fragment C; UreB: Urease B; WSSV: White spot syndrome virus.

Antigen description

Target pathogen

Table 1. Summary of Bacillus subtilis-based vaccines (cont.).

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Bacillus subtilis comes of age as a vaccine production host and delivery vehicle

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doi: 10.1586/14760584.2015.1051469

doi: 10.1586/14760584.2015.1051469

Tetanus toxin fragment C

Multiple epitopes of foot-and-mouth disease virus (TEpiAs) and cholera toxin B subunit

Group A bovine or murine rotavirus VP6

26 kDa fulllength GST protein

2B subunit of Shiga-like toxin (Stx2B)

C. tetani

Foot-and-mouth disease virus

Rotavirus

Schistosoma japonicum

Enterohemorrhagic E. coli O157:H7

[62]

[65]

[66]

[71]

[73]

BALB/c mice were orally immunized with 1 X1010CFU of vegetative cells) on days 1–3 and 21–23. Guinea pigs were orally immunized with 5X1010 CFU of vegetative cells on days 1–3 and 21–23. IgG in serum and IgA in lung and gut lavage fluid were induced; lymphocyte proliferation and antigen specific-IFN-gamma responses of T lymphocytes were detected. Challenge experiment with mean infective dose (ID50) of Foot-and-mouth disease virus revealed a protection rate of 60% Mice were immunized by the intranasal route on days 0, 14 and 21 with spores or vegetative cells along with CT or a mutant form (R192G) of E. coli heat-labile toxin (mLT) as adjuvants. Serum anti-VP6 IgG responses were induced in both spore- and vegetative cells-treated groups, but only mice immunized with spores induced anti-VP6 IgA responses. Mice were virus challenged 3 weeks after the last immunization and diminished virus shedding was observed in the spores/CT or mLT-treated group Mice were immunized by the oral route with spores on days 1, 2, 3, 17, 18, 19, 34, 35 and 36. Both seric IgG and intestinal IgA antibody responses were elicited but immunoprotection was not evaluated Mice were immunized by the oral, nasal or subcutaneous routes with either vegetative cells or spores. Vegetative cells by the oral route induced low anti-Stx2B serum IgG and fecal IgA responses while mice immunized with recombinant spores developed antiStx2B responses only after administration via the parenteral route. Nonetheless, the elicited responses were not sufficient to neutralize the toxin in in vitro and in vivo assays

Secretory expression of TEpiAs fused to cholera toxin B under control of promoter p43 of B. subtilis

Intracellular expression of VP6 under the control of the Pspacp1/2 constitutive promoter in B. subtilis vegetative cells or spores

GST coding gene (SjGST) was fused to full-length CotC gene and expressed in spores

Full-length Stx2B gene was expressed under the control of a stress inducible promoter (sigma B-dependent promoter derived from the B. subtilis gsiB gene)

Ref.

Mice were immunized by the intranasal route on days 0, 14 and 28. Immunized mice generated protective systemic and mucosal antibodies and survived challenge with 2 LD100 of tetanus toxin. Isotype analysis of serum antibody indicated a balanced Th1/Th2 response

Immunogenic properties

B. subtilis strains expressing TTFC on the surface of spores through fusion with CotC or in cytosol of vegetative cells through transcriptional fusion were obtained

Expression approach

Several pathogens have been targeted through B. subtilis-based formulations leading to relevant outcomes in preclinical evaluations. BCG: Bacille Calmette–Guerin; CFA: Colonization factor antigen; CFU: Colony forming unit; CsLAP2: C. sinensis Leucine aminopeptidase; CT: Cholera toxin; ETEC: Enterotoxigenic E. coli; GST: Glutathione-S-transferase; IFN: Interferon; MPT: Mycobacterium tuberculosis; TTFC: Tetanus toxin fragment C; UreB: Urease B; WSSV: White spot syndrome virus.

Antigen description

Target pathogen

Table 1. Summary of Bacillus subtilis-based vaccines (cont.).

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Expert Rev. Vaccines

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A carboxyterminal segment of the alpha toxin gene (cpa) fused to the GST gene, named GSTCpa247-370

Anthrax protective antigen

CfaB from the CFA/I fimbriae

Clostridium perfringens

Bacillus anthracis

Enterotoxigenic E. coli

[68]

[69]

Mice were immunized by the intraperitoneal route on days 0, 14 and 28. Spores from all the antigenic strains induced serum anti-PA IgG responses. Spores displaying only domain 1b-3 generated lower IgG responses compared to those expressing domain 1b-3 plus the full-length PA or only full-length PA (in germinated spore). Co-expression of PA on the spore coat and in the vegetative cells (germinated spore) increased the neutralizing antibody levels measured in an in vitro toxin neutralization assay as well as full immunoprotection after intraperitoneal challenge with B. anthracis spores

Mice were primed with pRECFA by the intramuscular route on days 0 and 14; four weeks later were subsequently boosted by the oral route with two oral doses, each consisting of three consecutive daily doses, of either vegetative cells or spores given in a 2-week interval Mice boosted with B. subtilis strains induced higher levels of antiCfaB antibodies with increased affinity for native CFA/I. Sera from these animals inhibited adhesion of ETEC cells to human red blood cells, and neutralized ETEC strain in a suckling newborn mice challenge model

Surface expression display, intracellular expression and combination of both was studied. PrrnO-driven intracellular expression while expression display driven by CotB and CotC endogenous promoters. Domain 1b-3 was fused to the C-terminus of CotB (DL374) and domain 4 was fused to a truncated CotB protein (DL387) or full-length CotC (DL372). In addition, strains expressed one of the three chimeric proteins on the spore coat (CotB-PA1b-3, CotB_-PA4, or CotC-PA4) as well as the full-length, secretable, form of PA in the vegetative cell (or germinated spore) were obtained A DNA vaccine (pRECFA) was used for priming, which encodes a membrane-anchored hybrid CfaB protein sandwiched fused with the herpes simplex virus type 1 glycoprotein D B. subtilis LDVanc3 strain was engineered to achieve surface expression display by the use of the signal sequence of the Bacillus amyloliquefaciens amyQ gene and the carboxy-terminus of the cell wall anchor domain of the Staphylococcus aureus fibronectin-binding protein (FnbB) B. subtilis LDV6 strain was engineered to achieve intracellular expression Expression was driven by the stress inducible gsiB gene promoter

[67]

Mice were immunized by the oral or intranasal routes with three doses of B. subtilis on days 1, 21 and 42. Recombinant spores expressing GST-Cpa247-370 showed the most potent responses in terms of IgG responses in sera and IgA responses in saliva, feces and lung. Neutralizing IgG antibodies were evidenced through in vitro and in vivo assays, and mice were protected against a toxin challenge

Three expression modalities were assessed: in the vegetative cell; on the surface of the spore coat through fusion to CotB and a combination of both types of expression in a single strain

Ref.

Immunogenic properties

Expression approach

Several pathogens have been targeted through B. subtilis-based formulations leading to relevant outcomes in preclinical evaluations. BCG: Bacille Calmette–Guerin; CFA: Colonization factor antigen; CFU: Colony forming unit; CsLAP2: C. sinensis Leucine aminopeptidase; CT: Cholera toxin; ETEC: Enterotoxigenic E. coli; GST: Glutathione-S-transferase; IFN: Interferon; MPT: Mycobacterium tuberculosis; TTFC: Tetanus toxin fragment C; UreB: Urease B; WSSV: White spot syndrome virus.

Antigen description

Target pathogen

Table 1. Summary of Bacillus subtilis-based vaccines (cont.).

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Bacillus subtilis comes of age as a vaccine production host and delivery vehicle

Review

doi: 10.1586/14760584.2015.1051469

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Rosales-Mendoza & Angulo

There are still some vaccines that require a detailed characterization. For example, candidates that remain to be evaluated in terms of immunoprotection include the vaccine candidates against Human papilloma virus type 33 [70] and Schistosoma japonicum [71]. Another is for C. difficile where only one candidate was evaluated but not characterized in terms of immunogenicity [72]. On the other hand, one vaccine against enterohemorrhagic E. coli O157:H7 failed to provide protective immunity [73].

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Induction of antibody-mediated responses

Humoral immune responses are of key importance to combat extracellular pathogens or prevent viral particles from entering the host cells. In fact, several commercial vaccines rely on the induction of antibody-mediated immunity. In the field of B. subtilis-based vaccines, mucosal and systemic immunoglobulins have been robustly induced by formulations delivered by either nasal, sublingual or oral routes. In this context, basal levels of sIgA and IgG secreted into the mucosal surface must play a pivotal role in recognizing (opsonization) B. subtilis or B. subtilis-displaying antigens, serving as a bridge for easier uptake through the antibody Fc domain and the Fc receptor of phagocytes and APCs. After prime-vaccination and boosts, the recruitment of B-cell clones producing specific antibodies toward the antigen is reinforced. Most of the reports have demonstrated the immunogenicity of B. subtilis-based vaccines in terms of the induction of mucosal and systemic antibody responses. In particular, in terms of immunoprotection, the induction of C. perfringens neutralizing IgG antibodies was achieved after oral B. subtilis-antigen immunization as evidenced in a mice model after administration of a LD100 toxin dose [67]. A potential protective mechanism could consist of host mucosal exclusion, since antibodies produced by B. subtilis-based vaccination are able to inhibit S. mutans adhesion in vitro [74]. Also, mucosal-specific sIgA production seems to be important for reducing H. pylori stomach load through an oral vaccine formulated with B. subtilis expressing the urease B antigen [55]. As for the less explored sublingual and intranasal administration routes, IgG and IgA in feces, saliva and vaginal fluids are strongly triggered after B. subtilis-tetanus toxin fragment C vaccination and even immunoprotection has been achieved against C. tetani toxin challenge in mice and piglets [60–63]. The proof-of-concept on protection against viral diseases using B. subtilis-based vaccines has been achieved for Foot-and-mouth disease virus and bovine A group Rotavirus, where specific mucosal and systemic antibodies correlated with the observed protection [65,66]. Considering that multicellular parasite infection generally induce Th2-polarized immune responses that promote B-cell activation and antibody production, the induction of humoral responses and immunoprotection was observed following an oral immunization scheme with B. subtilis-expressing antigens from C. sinensis and S. japonicum [59,71]. Considering all together, B. subtilisbased vaccines have a remarkable potential for the induction of antibody-mediated immune responses and protective immunity against infectious diseases. doi: 10.1586/14760584.2015.1051469

Induction of cellular-mediated responses

One aspect that deserves special consideration is the induction of cytotoxic T lymphocyte (CTL) responses because of their critical importance in the fight against intracellular pathogens and cancer. Therefore, Th1 responses are a critical goal to address for many vaccination strategies. However, to date most of the soluble antigen-based subunit vaccines have failed to induce robust CTL responses as the antigen presentation is biased to the MHC class II pathway that may lead to humoralpolarized immune response (Th2) instead of cellular-immune response (Th1-CTL), which typically requires antigen processing via endogenous pathways before presentation on the cell surface in association with MHC class I molecules [75]. Thus, in this case CTL responses are not induced unless particular adjuvants are co-administered. With this in mind and given that a weakness of many adjuvants is their lack of induction of CTL responses [76], lipid- and polymer-based particulate antigen delivery systems have been developed showing promising results. However, the elevated cost constitutes a major limitation on the formulation of these vaccines. Some alternatives using whole cell vaccines have been developed. For example, the yeast Saccharomyces cerevisiae has been proposed as a convenient vector for the elicitation of CTL responses. Yeast strains expressing tumor or HIV-1 antigens were used to elicit potent antigen-specific CTL responses in test animals. In accordance with this finding, it has been reported that yeast induces dendritic cell maturation, IL-12 production and an efficient priming of MHC class I- and class II-restricted T-cell responses [77]. In this context, it is remarkable that cellular responses have been successfully elicited by B. subtilis-based vaccines as has been proven for candidate vaccines targeting H. pylori [55], Mycobacterium tuberculosis [53], Human papilloma virus [70] and Foot-and-mouth disease virus [65]. These cellular responses were achieved using B. subtilis strains expressing the antigen under either intracellular or spore display strategies, which were administered by mucosal routes. In the case of vaccine candidates against S. mutans [74] and C. tetani [60–63], the fact that sublingual administration was found highly effective on inducing strong humoral and cellular responses deserves special interest. This latter is in agreement with findings that have been reported during the last 5 years for different vaccination models comprising soluble proteins, inert particulate antigens as well as live attenuated viruses [78]. The overall findings indicate that both systemic and mucosal immune responses, including B-cell (antibody) and T-cell responses, have been successfully induced at mucosal and systemic levels. The efficacy of this sublingual administration is comparable to intranasal antigen administration in terms of magnitude, breadth and anatomic dissemination [60,62,63]. Remarkably, when the antigen is administered by the sublingual route, the vaccine components are not redirected to the brain, as it occurs by the intranasal route which can affect vaccine safety. The better immune response to sublingual vaccination might be ascribed to the fact that sublingual delivery has a smaller distribution volume and a less aggressive environment Expert Rev. Vaccines

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Bacillus subtilis comes of age as a vaccine production host and delivery vehicle

than the gastrointestinal delivery [79]. However, in the case of the B. subtilis-based vaccines, sublingual administration has received little attention, which highlights the need for exploring further target antigens following this approach as a field of opportunity to possibly achieve highly effective immunizations. Of particular interest is a recent report by Hinc et al. [54], where recombinant spores presenting IL-2 were evaluated as a co-administered adjuvant along with spores expressing the target antigen, a fragment of subunit B of Helicobacter acinonychis urease, fused to the coat protein CotC as well as the full-length ureB gene of H. acinonychis under control of the promoter of rrnOP operon to achieve expression in vegetative cells. In this study, the goal was to impose a strong induction and polarization toward cellular immune responses of high relevance in the fight of a myriad of pathologies. Remarkably strong cellular immune responses were elicited when the combination of both spores expressing IL-2 and urease B were administered to mice by the oral route. The outcomes identified in the field of B. subtilis-based vaccines in terms of cellular immunity reflect a substantial potential for the development of vaccines against viral and other intracellular pathogens. As an example relevant pathogens, such as HIV, remain to be studied under this focus as a convenient immunization approach [80]. For influenza, another relevant disease globally spread, only one group following the strategy based on antigen adsorbed on spores has been explored [52]. Several efforts to develop vaccines against chronic non-communicable diseases are ongoing [81–83]. In fact, a vaccine prototype produced on a recombinant strain of Lactococcus lactis has been reported [84]. Since no vaccination prototypes against noncommunicable diseases have been reported using B. subtilis, this constitutes an important field to be explored with the potential to yield convenient approaches. Type I diabetes, hypertension and cancer are high impact pathologies that could be addressed through B. subtilis-based vaccines. Moreover, neglected tropical diseases should be studied from the viewpoint of using B. subtilis-based vaccines to fight against these diseases [85]. Five-year view

Since the first publication describing the use of B. subtilis spores as a vaccine delivery system in 1997 and the first patent for this technology in 1998 [86,87], the capability of B. subtilisbased vaccines for inducing strong immunoprotective responses in several vaccination approaches at the preclinical level has

Review

been well proven. These vaccination approaches are attractive in terms of costs since complex purification steps, cold-chain for distribution and adjuvants requirement may be avoided. The identified short-term goals for the field include safety evaluations at the preclinical level to facilitate progression into clinical trials for B. subtilis-based vaccine development. The last report from WHO reflects that people are infected with HIV/ AIDS (35 million), TB (9 million) and influenza (5 million), all of them are high epidemiologic impact diseases to which efficacious vaccines are urgently needed, especially for developing countries [4]. Essentially these diseases have not been targeted through the use of B. subtilis-based vaccines, thus we propose this as a biotechnological option for future research. The evidences on the induction of antibody- and cellmediated immunity makes B. subtilis-based vaccines of remarkable importance since the induction of such responses are a challenge when subunit vaccines are used. In addition, the expression of distinct cytokines would open new alternatives to enhance or achieve specific immune polarization. The expression and evaluation of IL-12 and IL-17 (Th17 immunity) would provide interesting insights on the potential of addressing shifts of immune cellular responses. Moreover, conducting a systematic polyfunctional T-cell analysis is proposed as a relevant goal in the characterization of B. subtilis-based vaccines. Cells producing several cytokines are considered potent inducers of protective immunity against viral infections and other intracellular pathogens [53]. New pending research efforts also comprise studies on antigen stability and dosage optimization to address cost-effective immunoprotection. In conclusion, B. subtilis-based vaccines represent an easy to produce, practical to handle, human-safe and an economically feasible opportunity to fight against diseases of human and animal populations. It is envisioned that the sustained research activity in this field will lead to new vaccines being evaluated at the clinical level in the next 5 years. Financial & competing interest’s disclosure

S Rosales-Mendoza was supported by CONACYT/Mexico CONACYT (grant INFR-2014-01-225843) and PROFOCIES 2014. CE Angulo was supported by CONACYT (grants CB-2010-01, 151818 and INFR-2014-01, 225924). 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.

Key issues .

Bacillus subtilis constitutes a vaccine biofactory and delivery vehicle of low production cost, straightforward administration and attractive immunogenic properties.

.

The technologies that allow for the design of B. subtilis-based vaccines are summarized.

.

An updated outlook of the pathogens targeted under this technology is provided.

.

Several vaccine candidates have been evaluated with promising findings in terms of the induction of effective humoral and cellular immunoprotective responses.

.

Perspectives for the development and evaluation of B. subtilis-based vaccines are identified.

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doi: 10.1586/14760584.2015.1051469

Expert Rev. Vaccines

Bacillus subtilis comes of age as a vaccine production host and delivery vehicle.

Bacillus subtilis is a vaccine production host and delivery vector with several advantages such as a low production cost, straightforward administrati...
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