The Potential of Precision Probiotic Hafnia alvei HA4597 to Support Weight Loss

Abstract

Hafnia alvei HA4597® is a novel probiotic strain producing an anorexigenic mimetic protein. This report summarizes the innovative approach leading to the discovery of the precision probiotic H. alvei HA4597® and its benefits on body weight and metabolic parameters. H. alvei HA4597® has been identified after the striking findings on the effects of the bacterial metabolite ClpB (Caseinolytic peptidase B) on appetite regulation, through a screening of ClpB-producing strains. Its efficacy in humans has been validated by a multicentric, double-blind, randomized placebo-controlled trial including 236 overweight adults. The successful results on body weight loss of the clinical study support the use of H. alvei HA4597® in the global management of excess weight.

1. Introduction

With the growing obesity epidemic and its associated comorbidities including cardiovascular diseases, diabetes, musculoskeletal disorders, cancer [1], depression [2, 3], and now even Covid-19 [4, 5], there is an urgent need to address this public health issue. As highlighted by the World Health Organization (WHO), obesity is preventable.

The understanding of the physiopathology of obesity started to shift back in 2006 when a publication in Nature highlighted that the microbiota of obese individuals differed from that of lean individuals. In addition, when this microbiota was transplanted into mice, propensity to gain weight was transmitted as well [6].

Since the discovery of the roles of the microbiota in metabolism, Pr. Sergueï Fetissov, neuroendocrinologist and professor of physiology, investigated extensively the roles of the gut microbiota in host appetite control. As early as 2002, he identified human autoantibodies reacting with the key hormone of satiety alpha-melanocyte stimulating hormone (alpha-MSH). He discovered an anorexigenic peptide produced by the microbiota with a homology of sequence with alpha-MSH [7]. In a review published in Nature Reviews Endocrinology, he demonstrated that metabolites produced by the microbiota can regulate food intake, and more specifically identified a bacterial protein called caseinolytic peptidase B (ClpB), a conformational antigen mimetic of alpha-MSH (Figure 1) [8, 9].

Figure 1.

Adapted from Sergueï Fetissov, nature reviews 2016.

Observations showed that ClpB levels in human fecal or serum samples negatively associated with Body Mass Index (BMI) [10]. Pr. Sergueï Fetissov and Pr. Pierre Déchelotte, gastroenterologist, professor in human nutrition and director of the Gut Brain Axis Laboratory at the French National Institute of Health and Medical Research (Inserm), carried on their investigations to better understand and describe the cause-and-effect relationship, demonstrate the proof of concept on animal models and translate the discovery into a lever of action in the battle against obesity.

2. From amino acid sequence homology to functional mimicry of the anorexigenic pathway

In the beginning, the bacterium encountered to study ClpB was E. coli K12, the standard model used in biotechnology. In silico sequence alignments allowed to identify a 6-amino acid sequence homology between a bacterial protein, E. coli K12 ClpB, and alpha-MSH [11]. The properties of the E. coli K12 ClpB sequence explain its folded shape, exposing the epitope and thus making it a specific conformational mimetic of alpha-MSH [9, 11].

The functional mimetic properties of E. coli K12 ClpB were first confirmed with a proteomic approach. ClpB was identified after Western Blot analysis of E. coli K12 stationary phase total protein extract of E. coli K12 using anti alpha-MSH IgG [9]. Then, two pathways used by the ClpB to regulate satiety were identified with preliminary protocols in mice:

• Both ClpB protein (recombinant) and E. coli K12 total stationary phase protein extract act locally in the gut by stimulating peptide YY (PYY) secretion by L-cells [12, 13, 14]. In addition, colonic infusion of E. coli K12 stationary phase protein extract on rats increases plasma PYY levels [13].

• ClpB also circulates in the blood and can stimulate the anorexigenic pathways in the hypothalamus by activating POMC neurons [13].

Anorexigenic properties of E. coli K12 ClpB were then confirmed in vivo on mice. Oral gavage of mice with E. coli K12 triggers a decrease of the food intake and body weight that wasn’t shown with ClpB-deficient strains [14].

The alpha-MSH mimetic properties of bacterial E. coli ClpB made it a good candidate to develop an anti-obesity solution, but to develop a product with unquestionable safety, the team looked for a ClpB-producing strain with a food grade status. Hafnia alvei, a starter culture used in cheese production, in particular the Normandy traditional cheeses Camembert and Brie, was identified thanks to in silico approach [14]. H. alvei is a commensal bacterium from the Enterobacteriaceae family, naturally present in cheese [15, 16] and in the human gut [14], and thus benefitting from a long history of safe use.

The summary of these steps of discovery and development are exposed in Figure 2 hereunder.

Figure 2.

From in vitro identification of ClpB to in vivo proof of concept.

3. Hafnia alvei HA4597® reduces body weight gain, fat mass and food intake in murine models of obesity

The first preclinical study was conducted to validate H. alvei and its production of ClpB, as a potential probiotic and its postbiotic for appetite and body weight management in overweight and obesity. Legrand et al. [14] tested H. alvei HA4597® on two mouse models of obesity: genetic, leptin deficient ob/ob mice (a model of hyperphagia) and nutritional, High-Fat Diet (HFD)-induced obesity.

This study confirmed that H. alvei HA4597® significantly reduces body weight gain and fat mass in both models of obese mice (Figure 3a-d). In the hyperphagic ob/ob model, H. alvei HA4597® treatment significantly reduces food intake with a 20.8% decrease (vs placebo) in total intake measured after 18 days (p < 0.001), the difference becoming significant from day 8 (Figure 3e) and was accompanied by a higher level of phosphorylated hormone-sensitive lipase in fat tissues (p < 0.01) (Figure 3g).

Figure 3.

Results of Hafnia alvei HA4597® supplementation vs. control in Ob/Ob mice and HFD mice models of obesity (adapted from Legrand et al., 2020). Body weight dynamics in ob/ob (a) and in HFD-fed obese mice (b), Two-way RM ANOVA, p < 0.001, (b) *for days 23–25, 38, 41, and 47, **for days 26, 27, 30, 37, 39, 42, and 43, ***for days 28, 29, 31–36, and 40. Total fat and lean tissue mass in ob/ob (c) and in HFD-fed obese mice (d). Mann–Whitney tests, **p < 0.01, *p < 0.05. Cumulative food intake in ob/ob (e) and in HFD-fed obese mice (f), Two-way RM ANOVA, p < 0.05, Bonferroni post tests, ***p < 0.001, **p < 0.01, *p < 0.05. Actin-normalized pHSL levels in the epididymal fat tissue in ob/ob (g), Mann-Whitney tests, *p<0.05.

The effect of H. alvei on food intake was not observed in mice on HFD, considering that HFD models tend to hypophagia because mice are not attracted to fat (Figure 3f).

Thus, H. alvei HA4597® exhibits the desired probiotic properties of an appetite and body weight management supplement i.e., it triggers anorexigenic and lipolytic effects in hyperphagic mice resulting in decreased body weight gain and fat mass.

A second trial conducted by Lucas et al. [17] evaluated the efficacy of Hafnia on another animal model of obesity based on a combined model of both HFD-fed and genetic ob/ob mice that may represent most closely hyperphagia and diet-induced obesity in humans (compulsive eating behavior combined with hypercaloric diet and accompanied by functional leptin resistance). This study also compares the efficacy of the strain to the drug Orlistat, a lipase inhibitor used in humans for the management of obesity.

A daily provision of 1.4 x 10¹⁰ Colony Forming Units (CFU) of H. alvei HA4597® in these mice significantly decreased the food intake, the body weight gain (Figure 4A and B) and total fat mass and preserved lean mass, resulting in an improved lean/fat mass ratio (Figure 4C). On top of that, other metabolic parameters were improved with the H. alvei treatment, including glycemia, total cholesterol and hepatic alanine aminotransferase (ALAT) (Figure 4E).

Figure 4.

Results on body weight gain and other parameters after 37 days of oral gavage with Hafnia alvei HA4597® vs. orlistat in HFD Ob/Ob mice (adapted from Lucas et al., 2020). (A) Cumulative food intake (g). Two≃way RM ANOVA, p < 0.0001, Bonferroni’s posttests, HFD vs. HFD + Orlistat. *** p < 0.001; ** p < 0.01 days 24,25; * p < 0.05 days 22,23. HFD + H. alvei vs. HFD + Orlistat. *** p < 0.001, ** p < 0.01, days 17,18; *p < 0.05 days 15,16. (B) Body weight gain (%). Two≃way RM ANOVA, p < 0.0001, Bonferroni’s posttests, HFD vs. HFD + Orlistat, *** p < 0.001,** p < 0.01, * p < 0.05; C. ANOVA, p < 0.0001, Tukey’s posttests avs. HFD and bvs. HFD + H. alvei, both *** p < 0.001, Student’s t≃tests, * p < 0.05, (mean ± SEM; SDiet, n = 16, all other groups, n = 24). (C) Lean/fat mass ratio at the end of the treatment. Student’s t≃tests, # p < 0.05, (mean ± SEM; SDiet, n = 16, all other groups, n = 24). (D) Basal glycemia at end of treatment (g/L). ANOVA p < 0.05, Tukey’s posttest vs. SDiet * p < 0.05. Student’s t≃test # p < 0.05. (E) Alanine transaminase (ALAT) levels at end of treatment (U/L). ANOVA p = 0.0006, Tukey’s posttests ** p < 0.01, * p < 0.05, (mean ± SEM; SDiet, n = 8, all other groups n = 12).

Although Orlistat is effective for weight loss, in contrast to H. alvei HA4597®, it is accompanied in this study by a strong hyperphagic effect which may be the cause of the increased glycemia observed for this group (Figure 4D).

After H. alvei proved successful in the regulation of appetite and weight in mice models, the next step was to evaluate efficacy in humans.

H. alvei HA4597® improves weight loss in a multicentric, double-blind, randomized placebo-controlled trial including 236 overweight adults.

To investigate the clinical efficacy of the strain, a 12-week prospective, double-blind, randomized study was realized on 236 overweight men and women (230 followed protocol and were analyzed in the per protocol results) [18]. All subjects were on a − 20% low-caloric diet and were asked to maintain their usual physical activity. Subjects received either 2 capsules per day providing 10¹¹ bacteria of H. alvei HA4597® per day (HA) or a placebo (P).

The primary outcome was the percentage of subjects losing 3% or more of their body weight after 12 weeks. Indeed, significantly more subjects (+38%) lost at least 3% of their initial weight after 12 weeks than in the placebo group (57.7 vs. 41.7%, p = 0.028, Per Protocol results) (Figure 5A). 51% more participants also lost more than 4% of their body weight on HA than on placebo (46.2% vs. 30.6%, p = 0.024) (Figure 5B). The average weight loss observed in the treated group was 3.6% at 12 weeks, vs. 2.9% in the placebo group, a high figure linked to the strong effect of the hypocaloric diet.

Figure 5.

Results of the clinical study with 12-week supplementation of overweight subjects with Hafnia alvei HA4597® or placebo (adapted from Déchelotte et al., 2021, except Figure 5D, unpublished). (A) Proportion of subjects who lost at least 3% of body weight after 12 weeks PP population, Exact Fisher’s test P. vs HA. *p≤0.05. (B) Proportion of subjects who lost at least 4% of body weight after 12 weeks PP population, Exact Fisher’s test P. vs HA. *p≤0.05. (C) Hip circumference change vs. T0 ITT population, Mann-Whitney-U test (w12-w0) P. vs. (w12-w0) HA. *pU≤0.001. (D) Serum glucose concentration before and after supplementation ITT population, Mann-Whitney-U test (w12) P. vs. (w12) HA. *pU ≤ 0.05. (E) Feeling of fullness ITT population, Mann-Whitney-U test (w12) P. vs. (w12) HA.**pU ≤ 0.01. (F) Change in Feeling of fullness ITT population, Mann-Whitney-U test; (w12-w0)P. vs. (w12-w0)HA.* pU ≤ 0.05. Paired Wilcoxon test; HA(w0) vs. HA(w12).** pwi ≤ 0.01.

Compared to the placebo group, the participants in the probiotic group saw a significant further 1.2 cm reduction in hip circumference (p < 0.001 at 12 weeks) as well as a significant decrease in blood glucose (p = 0.027 at 12 weeks) (Figure 5C and D). Furthermore, the HA group recorded a significant decrease of cholesterol compared to the beginning of the study (p = 0.008 for total cholesterol and p = 0.028 for LDL-cholesterol at 12 weeks) (unpublished).

Importantly, this study also revealed an increased feeling of fullness assessed by visual analog scale (VAS) (p = 0.009 at 8 weeks) in the HA group (Figure 5F). In the VAS, a score of 50 means that there is no feeling of hunger. Both groups were under a − 20% hypocaloric diet, but only the treated group led to feeling of fullness scores above 50 (Figure 5E). This confirms the mechanism of action of this precision probiotic through the regulation of appetite and this led, despite the constraints of the diet, to a significantly higher level of satisfaction of the subjects from the treated group, compared to the placebo group.

Indeed, in the HA group, benefit of treatment was rated as “very good” or “good” by 67.9% of subjects compared to 53.1% in the placebo group (p = 0.019). Only 5% of subjects in the probiotic group rated it as “poor”, as opposed to 14.2% in the placebo group.

This level of satisfaction shows the users have a clear perception of efficacy, and the good appetite regulation during a diet makes it easier to keep compliant to the reduced caloric intake, without the difficulty and discomfort associated with hunger.

According to the official guidelines for the management of obesity, a 3 to 5% weight loss is the reference interval associated with meaningful clinical benefits: reductions in serum triglycerides concentrations, of blood glucose, HbA1C, and the risks of developing type 2 diabetes [19].

With clinically relevant efficacy as soon as in the first 12 weeks, excellent tolerability and no adverse events, these results support the use of H. alvei HA4597® in the global management of excess weight.

4. From the clinical setting to the market: benefits of H. alvei HA4597® for overweight to obese consumers

As previously described, H. alvei HA4597® through its production of ClpB, a mimetic of the anorexigenic hormone alpha-MSH, constitutes an innovative and effective solution for body weight management in overweight and obesity. The French biotech TargEDys, pioneer in microbiome-based solutions and grounded in 15 years of academic research in collaboration with the prestigious laboratories of Inserm (Institut national de la santé et de la recherche médicale) in Rouen, developed a food supplement with 100 billion cells of H. alvei HA4597® per daily dose. It is the first PreciBiomic Strain product on the market for body weight management combining efficacy with no side effects. During product development, TargEDys ensured that the strain produces ClpB during the fermentation process so that both the strain and its protein ClpB are present in the product.

A consumer study confirmed the efficacy of the product in overweight people, and even in people suffering from obesity, in real life conditions, i.e., without caloric restriction. Above all, this study confirmed that the extension of the program over time intensifies the body weight loss.

5. Conclusion

As concluded by the authors of the clinical trial, supplementation with the precision probiotic [20] H. alvei HA4597® represents an innovative and well-tolerated strategy to enhance the efficacy of dietary advice for the control of excess body weight; paving the way to precision nutrition thanks to a gut microbial-based personalized approach.

Acknowledgments

The authors join to thank all the people who made this research possible, from the researchers themselves, to the investors and funds, as well as all the people and institutions who contributed to all the articles cited in the list of references. They all participated to the development of this new solution, directly or indirectly.

Thank you most particularly to Pierre Déchelotte and Sergueï Fetissov who made the discovery of the conformational antigen ClpB and its role in appetite regulation and founded TargEDys to transpose this knowledge into a product and approach to safely help people regulate their hunger and weight.

Thank you to Manon Dominique, Marie Galmiche Nicolas Lucas et Romain Legrand for all their research work in investigating the precise mechanism of action of H. alvei and ClpB as Ph.D. students and TargEDys research team.

The authors also thank warmly all TargEDys employees and partners who worked together and enabled the development of processes and production of the strain, from fermentation to blending and encapsulation, all the suppliers who provide high-quality delivery systems and packaging, to optimize the protection of the bacteria and protein all the way to the gut as well as the product’s stability, and all the regulatory advisers that made it possible to reach the market with this food grade strain that was never used as a probiotic before.

Conflict of interest

The co-authors are employees of TargEDys, the company producing and commercializing a food supplement based on H. alvei HA4597.