Use of Traditional and Genetically Modified Probiotics in Human Health: What Does the Future Hold?

ABSTRACT

Probiotics are live, nonpathogenic microorganisms that confer benefits to human health when administered in adequate amounts. Among the frequent proposed health benefits attributed to probiotics, their ability to interact with the host immune system is now well demonstrated. Although history has revealed that probiotics were part of fermented foods in the past, clinicians have started to use them therapeutically in regular diets. Moreover, the use of genetically modified probiotics to deliver molecules of therapeutic interest is gaining importance as an extension of the probiotic concept. This chapter summarizes some of the recent findings and perspectives on the use of both traditional and genetically modified probiotics to treat human diseases as well as what the future may hold concerning the use of these probiotics in humans.

INTRODUCTION

Advances in recombinant technology (e.g., genetic engineering) and in the understanding of the human immune system have led to prodigious advances in the development of novel delivery systems for mucosal administration (1, 2). The administration of therapeutic molecules through mucosal routes offers several important advantages over conventional strategies (i.e., systemic injection) such as reduction of secondary effects, easy administration, and the possibility to modulate both systemic and mucosal immune responses (3). Moreover, it is important for molecules of health interest that exert their effects at mucosal surfaces, the gastrointestinal tract (GIT), for example, to be delivered directly to the appropriate site. Nonetheless, a major disadvantage of the mucosal route of administration is that the actual amount of protein to be administered needs to be large due to the very small quantities of protein that survive degradation at mucosal surfaces such as the GIT (1, 3).

It is becoming increasingly apparent that alternative approaches to conventional mucosal delivery systems (e.g., inert systems such as liposomes or nanoparticles and live attenuated bacterial or viral vectors) are required to control diseases in humans in the 21st century. The design of novel approaches combining genetic engineering and probiotic bacteria that allow precise targeting of molecules of health interest to the mucosa can represent an attractive alternative to attenuated pathogenic vectors (1, 4). Hence, the generation and use of such genetically modified probiotics (GMPs), expressing therapeutic molecules, can offer the opportunity to further investigate their effects for food, nutrition, environment, and health.

PROBIOTICS AND HUMAN HEALTH

The word “probiotic” is derived from Greek and means “for life” and was introduced in 1953 by Werner Kollath. Probiotics are defined as “live microorganisms that, when administered in adequate amounts, confer a health benefit on the host” (reference 5, p 11). The probiotic concept was born in the beginning of the 20th century (1906) when Henry Tissier (a French pediatrician at the Pasteur Institute, Paris) reported clinical benefits in treating diarrhea in children with bifidobacteria (initially called Bacillus bifidus communis), a dominant genus in the gut microbiota of breast-fed babies (6). The claimed effect was a displacement of pathogenic bacteria by bifidobacteria (6). Then, Elie Metchnikoff (a Russian microbiologist who received the Nobel Prize in Physiology or Medicine in 1908) issued the following hypothesis: the longevity of Bulgarians is associated with their high consumption of fermented foods, and particularly with the live lactic acid bacteria (LAB) they contain (6). Two major concepts arose from his observations: (i) live bacteria that interact with humans can have beneficial effects, and (ii) these bacteria and their host have developed sophisticated cross-talk strategies which allow them to combine efforts to maintain or restore host health. Finally, in 1965, Lilly and Stilwell proposed the use of probiotics to enhance intestinal health (6).

Today, numerous bacteria are used as probiotics, and the most common strains belong to Bifidobacterium and Lactobacillus spp. Some species of these genera are natural inhabitants of the GIT, where they favorably influence intestinal microbiota homeostasis by inhibiting growth of harmful bacteria, maintaining a good epithelial barrier homeostasis, and promoting efficient food digestion, among other beneficial effects (7). However, commercial abuse of the term “probiotic” has become a major issue, with many products exploiting the term without meeting the requisite criteria. Moreover, probiotic products have received the legitimate attention of regulatory authorities that have an interest in protecting consumers from misleading claims. So far, the only approved probiotic claim by the European Food Safety Authority (EFSA) currently relates to improved lactose digestion through the action of the yogurt starters Lactobacillus delbrueckii subsp. bulgaricus and Streptococcus salivarius subsp. thermophilus (8). Indeed, many people who have congenital lactase (the enzyme that degrades lactose) deficiency do not tolerate this sugar (which is abundant in milk products). Clinical manifestations include diarrhea, abdominal colic, and flatulence. Interestingly, these symptoms appear with milk ingestion but are basically absent when yogurt is ingested. Thus, the EFSA has approved a health claim for L. delbrueckii subsp. bulgaricus and S. salivarius subsp. thermophilus in helping lactose digestion (8). This claim has been accepted because the mode of action is clearly understood and is due to the bacterial production of β-galactosidase, which degrades lactose in both the yogurt and in the gut. However, with the advances of the technology and the “omics” era (e.g., metagenomics, metabolomics, proteomics, etc.), as well as with the establishment of well-controlled pre- and clinical trials, novel probiotic claims (other than lactose digestion) will certainly be approved in the next few years. In the following paragraphs, we will briefly describe some beneficial effects of different probiotic strains to treat human diseases, particularly gastrointestinal disorders.

An important use of probiotics is the protection against pathogens. Indeed, probiotics might function as a physical barrier, impeding the colonization of the GIT by pathogenic bacteria. The most notable results have been obtained with some species of lactobacilli and bifidobacteria, which have been found to be particularly effective in treating diarrhea in newborns (9). Modulation of host immunity and promotion of host defense are the other most commonly supported benefits of probiotic consumption (7). The probiotic preparation VSL#3, which contains a mixture of eight bacteria, including lactobacilli and bifidobacteria species, has been shown to display anti-inflammatory properties, as well as protective effects in a murine model of intestinal inflammation (10). In this context, human clinical trial results have confirmed various therapeutic effects of such selected strains of probiotics in inflammatory bowel diseases (IBDs), notably VSL#3 in pouchitis patients (11), Escherichia coli Nissle 1917 in ulcerative colitis patients (12), and Lactobacillus salivarius UCC4331 and Bifidobacterium infantis 35624 strains in irritable bowel syndrome (IBS) (13). Oral administration of a strain of Lactobacillus plantarum to interleukin (IL)-10 knockout mice attenuates the severity of the spontaneous colitis in these mice (14). Recent studies in preclinical murine models have also shown that Lactobacillus casei BL23 can stimulate systemic immunity and protect against intestinal inflammation (20) as well as colorectal cancer (15). Bifidobacterium animalis subsp. lactis CNCM I-2494, a probiotic strain with a long background of use in fermented dairy products (16), was able to restore gut barrier permeability in gut inflammation (17). Finally, L. casei has been found to be capable of stimulating an immune response in children who take an oral vaccine against rotavirus, which causes acute diarrhea in infants and young children in developing countries (18).

Probiotics can also act by reducing oxidative stress, which is characterized by an uncontrolled increase in the concentration of reactive oxidative species in the GIT, a phenomenon frequently observed in IBD patients (19). Hence, some studies have shown the potential effect of different probiotic strains in the treatment of IBD using a variety of animal models (20–22). Also, Lactobacillus rhamnosus CNCM I-3690, selected for its antioxidant properties in vitro (23), has shown anti-inflammatory activities in a colitis murine model as well as protective effects to induced barrier hyperpermeability in mice (24).

Altogether, these results confirm the potential of probiotics to regulate the host immune system and suggest complex cross-talk between the host and bacteria.

USE OF GMPs TO PREVENT AND TO TREAT HUMAN DISEASES

Considering the need to develop novel effective strategies for the delivery of therapeutic molecules at mucosal surfaces, Gram-positive LAB have emerged as attractive vehicles for the oral, intranasal, vaginal, and rectal delivery of such molecules at the end of the 20th century. Indeed, the potential of these bacteria to act as live mucosal vehicles, in particular, food-grade Lactococcus lactis strains, has been intensively investigated in the past 2 decades (25). Strikingly, these GM lactococci have been successfully used as vectors to deliver functional proteins at the mucosal level in preclinical studies using murine models, reproducing different human pathologies such as IBD, IBS, obesity, diabetes, and cancer (1). Since probiotic therapy is mainly focused on modulation of host immunity and promotion of host defense (see above), we can assume that GMPs are potential attractive candidates to deliver molecules of therapeutic interest to mucosal surfaces to prevent and to treat human diseases. Interestingly, the administration of such GMPs would allow a significant reduction in the treatments’ costs, a reduction of secondary effects, easy administration, and the possibility to modulate both systemic and mucosal immune responses. Oral administration is definitely the most common and convenient route of drug administration because of its simplicity, noninvasive nature, and low discomfort levels for patients. In addition, this route displays efficient therapeutic effects for certain disorders of the GIT such as IBD or IBS.

As previously stated, L. lactis has been the LAB most widely genetically engineered for the production and delivery of therapeutic molecules thus far. In addition, phase I and II clinical trials using GM strains of L. lactis secreting either IL-10 (26) or TTF (27) to treat Crohn’s disease and mucositis patients, respectively, have opened up new horizons in the use of GMPs in humans. Unfortunately, despite all the considerable and impressive work done using L. lactis as a live delivery vector, this bacterium has some drawbacks because of its very short survival time in the GIT and its lack of intrinsic immune-modulatory properties in contrast to lactobacilli which can persist longer in the GIT and possess interesting immune-modulatory properties such as anti-inflammatory activities (20, 28); these properties could enhance the anti-inflammatory potential of a GM strain in the context of IBD therapy, or proinflammatory activities, an interesting feature that could be exploited in the context of vaccination against a pathogen (e.g., adjuvant effect) (28). Thus, two other genera which have been subjected to recent investigations for heterologous expression of proteins of medical interest are Bifidobacterium and Lactobacillus spp. Indeed, the use of GM lactobacilli or bifidobacteria to produce and deliver recombinant proteins is an interesting and growing field of research, since these genera present several advantages compared to L. lactis when used as live mucosal vehicles, such as an increased persistence in the GIT and the immune-modulatory properties of some strains.

As mentioned previously, IBD is frequently associated with oxidative stress and epithelial damage; hence, GMPs delivering antioxidant enzymes can be an attractive preventive and therapeutic tool. Therefore, one strategy to prevent and treat intestinal inflammation is the use of GMPs to deliver antioxidant enzymes such as catalase, glutathione peroxidase, glutathione reductase, glutathione-S-transferase, or superoxide dismutase (SOD), which may be able to reduce reactive oxidative species concentrations in the GIT. A GM strain of Lactobacillus gasseri that produces SOD has been shown to exhibit anti-inflammatory effects in an IL-10-deficient mouse colitis model (29). In addition, GM strains of L. casei BL23 that produce either SOD or a manganese-dependent catalase were shown to display protective effects against reactive oxidative species and intestinal inflammation in mice (20, 30). Another well-designed study demonstrated that a mix of two GM strains of Streptococcus thermophilus CRL807 (a bacterium previously used as a starter to prepare a yogurt) with anti-inflammatory and anticancer properties, producing either catalase or SOD enzymes, displays higher anti-inflammatory effects than the wild-type counterpart strain in a murine model of colitis (31).

Recent studies have shown the important role of proteases and their endogenous inhibitors in the pathology of IBD (32). In this context, oral administration of a GM strain of L. casei BL23 that produces elafin, an endogenous protease inhibitor found in the human gut (33), prevents inflammation, accelerates mucosal healing, and restores colon homeostasis in different colitis models in mice (33). These encouraging results suggest that there may be a potential clinical application of GMP delivering elafin for IBD prevention and treatment in the future.

Bifidobacterium is another genus recently exploited as a GMP for drug delivery. The preliminary studies reporting the use of GM Bifidobacterium spp. to deliver drugs were by intravenous or systemic administration, rather than oral administration, to treat solid tumors in different animal models (34). The use of these methods was undoubtedly due to the ability of this genus to migrate from vascularized sites toward the tumors. This selective migration was attributed to the preference of Bifidobacterium spp. for anaerobic sites such as the hypoxic microenvironment found in solid tumors (34). It was not until a few years ago that the ability of a GM Bifidobacterium longum strain to deliver an anti-inflammatory molecule (i.e., IL-10 cytokine) was evaluated after oral administration in inflamed mice (35, 36). More recently, a study showed that oral administration with a GM B. longum strain expressing alpha-melanocyte-stimulating hormone (α-MSH) is efficient to treat colitis in mice (46). These results suggest an alternative approach to treat IBD by using GMP as a live vector to deliver α-MSH.

IS THERE A NEED FOR THE USE OF GMPs IN HUMANS?

The case of the anti-inflammatory IL-10 for the treatment of IBD, a common denominator for diseases such as Crohn’s disease and ulcerative colitis, is illustrative of the real need for the use of GMP in humans. IBD is a damaging chronic intestinal inflammation caused by a breach of tolerance for intestinal microbiota. IBD requires lifelong medication, having both fundamental medical as well as economic consequences. To make IBD treatment work, it is crucial to develop cheap and easy-to-administer therapeutics that are devoid of side effects. IL-10 was initially considered a good candidate for such IBD therapy. However, when IL-10 is applied by injection, side effects are induced that impede long-term use at elevated concentrations (37). The use of GM L. lactis for localized IL-10 synthesis may circumvent these fundamental obstacles. Delivery at the intestine of IL-10 by GM L. lactis treats or prevents IBD in mice (26).

In the same vein, intravenous administration with anti-tumor necrosis factor alpha (TNF-α) antibodies (e.g., infliximab, a human-murine chimeric monoclonal antibody that blocks the action of TNF-α) proved to be a breakthrough for IBD patients. However, although repetitive administrations of these antibodies can be effective, the treatment is costly, and it can be complicated by loss of response and associated with side effects (38, 39). Because several of these undesirable outcomes are associated with systemic application (i.e., intravenous injection), they might be solved by the use of a GM microorganism for local delivery of anti-TNF-α antibodies (40) (as for IL-10 cytokine).

Altogether, these studies show the interest in the use of GM L. lactis strains for local delivery of therapeutic molecules, and although there is no scientific evidence to support that these GM microorganisms are dangerous for human administration, it is indispensable to clearly demonstrate that it is safe to use such GMP strains.

CONCLUSIONS AND PERSPECTIVES

The results obtained in a phase I clinical trial conducted with a recombinant strain of L. lactis that produces IL-10 cytokine (see above and reference 41) revealed not only that the containment strategy used to construct this recombinant strain was safe and effective, but that local delivery of IL-10 to mucosal surfaces by a GM organism is also feasible in humans (42). Moreover, a phase IIa clinical trial in patients suffering from Crohn’s disease confirmed that the main primary endpoints of the study using this GM L. lactis strain expressing human IL-10 (named AG011) were achieved: safety and tolerability of the recombinant strain, environmental containment of the GM organism, and assessment of biomarkers associated with the strain. Unfortunately, concerning the endpoints of the evolution of the disease, the clinical results did not reveal a statistically significant difference in mucosal healing compared to the placebo group. Thus, we can envisage how ongoing work in different areas will help to improve the use of GM strains on human health, such as the use of more persistent LAB species (e.g., lactobacilli), the expression system to increase the quantities of the molecule delivered in situ, and the use of combinations of recombinant strains producing different types of therapeutic molecules (for review see reference 1). Such strategies should be tested in human clinical trials. Hence, a new phase Ib clinical trial using a GM strain of L. lactis expressing another therapeutic molecule, the human trefoil factor 1 (27), named AG013, showed that this GM organism was safe and well tolerated in patients with oral mucositis, an important inflammation and ulceration of the membranes covering the oral cavity, throat, and esophagus and among the most commonly reported adverse events associated with cancer chemotherapy. Strikingly, preliminary data demonstrated positive efficacy of this GM strain of L. lactis against oral mucositis in 25 patients compared to placebo (43).

In addition, a few challenges remain before potentially using GMP that expresses human elafin (e.g., recombinant lactobacilli strains) (33, 44) in clinical studies (P. Langella, personal communication). Certainly, elafin has been shown to be safe when delivered to humans (45), and GMPs have also been shown to be safe when given orally to humans (42). However, the safety of elafin-expressing GMP in humans remains to be tested, and a human clinical trial to address safety concerns will be necessary.

There is currently a large body of data in preclinical models to support the potential use of GM microorganisms as new therapies for human diseases (in particular, IBD). However, there is still a long way to go to reach the market for human use since important safety and regulatory issues still need to be addressed in depth.