NUTRACEUTICAL COMPOSITIONS

FIELD OF THE INVENTION

Nutrient formulations that include prebiotics, probiotics, and/or synbiotics and their use as nutritive oral adjuvants for enhancing immune response to vaccines in a subject.

NAMES OF PARTIES TO A JOINT RESEARCH AGREEMENT

The invention described herein is the subject of a Research Collaboration Agreement between GlaxoSmithKline Biologicals SA and the Washington University in St Louis entered into on Dec. 15, 2015, and amended Sep. 18, 2017, Jul. 1, 2018, Nov. 1, 2018 and Aug. 1, 2019.

BACKGROUND TO THE INVENTION

More than 100 million under-five children in the world are undernourished (FAO 2013 Hunger Report). The first 1000 days of a child's life disproportionately impact survival and immune development. Studies have shown that improving nutrition favorably increases immune development (Karacabey K, Ozdemir N (2012). The Effect of Nutritional Elements on the Immune System. J Obes Wt Loss Ther 2:152. doi:10.4172/2165-7904.1000152). Children in developing countries with high rates of enteropathogen exposure and undernutrition often exhibit poor responses to oral vaccination. Strategies aimed at improving vaccine efficacy in these vulnerable populations have had limited success. Oral vaccination schedules begin early in postnatal life when the gut microbiota, which plays a key role in mucosal immune system development, is undergoing maturation to an adult-like configuration. Perturbed microbiota development has been causally linked to undernutrition, raising the possibility that this perturbation contributes to impaired oral vaccine performance. Work has been done to provide food supplements to address malnutrition, but the idea of using nutrition to repair the gut microbiota and improve responses to vaccines, particularly orally administered vaccines, is an emerging concept.

Spirulina (Arthrospira genus, formerly classified in the Spirulina genus) is a filamentous microscopic blue-green algae (cyanobacteria) that forms tangled masses in warm alkaline lakes in Africa and Central and South America and is cultivated worldwide. The two most commonly utilized species of Spirulina are Arthrospira platensis and Arthrospira maxima. Spirulina has been shown to be a hepatoprotective agent (Jeyaprakash, K. and P. Chinnaswamy, 2005. Effect of Spirulina and Liv-52 on cadmium induced toxicity in albino rats. Indian J. Exp. Biol., 43: 773-781.), neuroprotective (Sharma, M. K., A. Sharma, A. Kumar and M. Kumar, 2007. Evaluation of protective efficacy of Spirulina fusiformis against mercury induced nephrotoxicity in Swiss albino mice. Food Chem. Toxicol., 45: 879-887), to be effective against inflammation (Coskun, Z. K., M. Kerem, N. Gurbuz, S. Omeroglu and H. Pasaoglu et al., 2011. The study of biochemical and histopathological effects of Spirulina in rats with TNBS-induced colitis. Bratislayske Lekarske Listy, 112: 235-243), to have antitumor activity (increase NK cytotoxicity—Akao, 2009; Nielsen, 2010; Hirahashi, 2002), antimicrobial activity (inhibit the growth of some Gram-negative and Gram-positive bacteria—Bhowmik, 2009), and to have antiviral activity against HIV (increase T helper lymphocytes (CD4+ counts) in HIV-infected patients accompanied by a decrease in the viral load—Teas, 2012; Ngo-Matip, 2015; Azabji-Kenfack, 2011), and also to provide antioxidant effects (Takeda, T., A. Yokota and S. Shigeoka, 1995. Resistance of photosynthesis to hydrogen peroxide in algae. Plant Cell Physiol., 36: 1089-1095).

Arthrospira plantensis is also used as a food source or dietary supplement in humans because it provides a quality source of protein in good quantity, provides essential amino acids, and provides minerals, vitamins and polyunsaturated fatty acids. Spirulina is considered a GRAS micro-organism (Generally Recognized as Safe) with no toxicity, and is approved as a food additive by the FDA (Food and Drug Administration) without risks to health (Parisi et al., 2009; Ambrosi et al., 2008). Several researchers (Miranda et al., 1998; Herrero et al., 2005; Souza et al., 2006; Mendiola et al., 2007; Bierhals et al., 2009) have reported the nutritional and functional importance of the compounds present in Spirulina platensis (spirulina).

Probiotics have been shown to improve digestion and to act as an adjuvant in immune therapy (Licciardi et al. Discov Med 2011; Maidens et al. Br J Clin Pharmacol 2013; Valdez et al. Trends Immunol 2014). Numerous species of bacteria have been found to provide positive effects and act as probiotics, including Bacillus sp., Bifidobacterium sp., Enterococcus sp., Lactobacillus sp., Lactococcus sp., Pediococcus sp., Saccharomycessp. and Streptococcussp. Such probiotic species are available marketed as dietary supplements and are available to consumers in powder, tablet, capsule, caplet, gel, beadlet, some in controlled release formulations, and in liquid form. Probiotics are also present in certain foods such as yogurt, sauerkraut, buttermilk, kefir, miso and juice, either naturally, or added as a supplement.

Encapsulation is also a way to protect bioactive agents from digestion in the stomach whereby the bioactive agent is encapsulated or coated with an emulsion or microemulsion. Such encapsulated bioactive agents show significant levels of absorption in the gastrointestinal tract, leading to increased bioactivity and bioavailability (Takahashi et al., 2007), and have high biocompatibility and versatility.

Several studies have demonstrated differences among strains of probiotic bacteria with regard to their survival in acidic environments. Probiotics must survive in gastric acids to reach the small intestine and colonize the host for appropriate prevention and management of several gastrointestinal diseases. To improve the survival rates of probiotic microorganisms during gastric transit, microencapsulation is considered to be a promising process. A variety of polymers may be used for microencapsulation but there is a need for improved physical and mechanical stability of the polymers used in probiotic microencapsulation. Toward that end, milk proteins have begun to be used in probiotic microencapsulation formulas.

Microencapsulation is a process in which the probiotic cells are incorporated into an encapsulating matrix or membrane that can protect the cells from degradation by the damaging factors in the environment and release at controlled rates under particular conditions. One purpose of microencapsulation of probiotics is to protect them from the low pH, bile salts, and other constituent products that are encountered during gastrointestinal transit. A microcapsule comprises a semipermeable or nonpermeable, spherical, thin and strong membrane surrounding a solid and/or liquid core with very small diameter, varying between a few microns and 1 mm. Desirable encapsulation materials for a nutraceutical include those generally recognized as safe (GRAS), i.e. ingredients that can be used in food preparation.

SUMMARY OF THE INVENTION

In an embodiment of the invention there is provided a nutraceutical composition comprising a probiotic species selected from Bacillus sp., Bifidobacterium sp., Enterococcus sp., Lactobacillus sp., Lactococcus sp., Propionibacterium sp., Pediococcus sp., Saccharomyces sp. and Streptococcus a Bacteroides, a Clostridioides, a Clostridium, an Erysipelotrichaceae, a Firmicutes, a Flavonifractor, a Fusobacterium, a Lactobacillus, a Parabacteroides, a Peptoclostridrium, a Robinsoniella, or a Subdoligranulum species; spirulina, a cereal and micronutrients.

One embodiment of the invention provides a nutraceutical composition comprising a probiotic species selected from a Bacteroides, a Clostridioides, a Clostridium, an Erysipelotrichaceae, a Firmicutes, a Flavonifractor, a Fusobacterium, a Lactobacillus, a Parabacteroides, a Peptoclostridrium, a Robinsoniella, or a Subdoligranulum species; spirulina, a cereal and micronutrients.

One embodiment of the invention provides a method of enhancing an immune response to a vaccine or antigen in a human, the method comprising administering an immune enhancing effective amount of a nutraceutical composition comprising: a probiotic species selected from Lactobacillus rhamnosus, Lactobacillus acidophilus; spirulina; cereal comprising flaxseed and amaranth; and micronutrients comprising i) vitamins B3, B6, C, D3, E and B9 and ii) minerals comprising magnesium, selenium and zinc; such that an enhanced immune response to a vaccine or antigen is observed in the human, as measured by an increase in mucosal IgA titer or as measured by an increase in systemic IgG titer.

One embodiment of the invention provides a method of using a probiotic to enhance the immune system in a human, the method comprising administering an immune enhancing effective amount of a probiotic species, wherein the probiotic species is a Bacteroides species, a Clostridioides species, Clostridium species, an Erysipelotrichaceae species, a Firmicutes species, a Flavonifractor species, a Fusobacterium species, a Lactobacillus species, a Parabacteroides species, a Peptoclostridrium species, a Robinsoniella species, a Subdoligranulum species or a combination thereof, such that the enhanced immune system is observable by an increased response to a vaccine or antigen in the human, as measured by an increase in mucosal IgA titer or as measured by an increase in systemic IgG titer.

One embodiment of the invention provides use of a nutraceutical composition as described herein for enhancing the immune system in a human by increasing an immune response to an antigen or a vaccine in said human, as measured by an increase in mucosal IgA titer or as measured by an increase in systemic IgG titer.

DESCRIPTION OF DRAWINGS/FIGURES

FIG. 1A and FIG. 1B show the designs of Study 1 and Study 2, respectively, of germ free mice receiving microbiota from undernourished human donors fed Probiotic (−) Nutraceutical Composition I (F4V formulation) in supplement of M18 diet followed by immunization with Cholera Toxin (CT)/OVAlbumin (OVA).

FIG. 2A and FIG. 2B show the results from Study 1 and Study 2 depicted in FIG. 1A and FIG. 1B, plotting fecal anti-CT IgA/Total IgA ratio (CT IgA ratio; Arbitrary Units) in mice receiving microbiota from different undernourished Donors.

FIG. 2C shows the anti-CT IgG responses measured by ELISA in serum obtained from Study 2.

FIG. 3A results of linear mixed-effects models showing the effects of microbiota and dietary supplementation on the CT-IgA ratio in feces from all mice colonized with the R and HypoR microbiota identified in Studies 1 and 2. *, P<0.05.

FIG. 3B shows the percentage of CD38^loGL7⁺ cells among CD19⁺TCRb⁻ cells in mesenteric lymph nodes (MLN) in mice in Studies 1 and 2 that were colonized with the R or HypoR microbiota and fed the M18 base diet or M18 supplemented with the Probiotic (−) Nutraceutical Composition I. P<0.05.

FIG. 3C is a heatmap showing the percent relative abundances of ASVs in mice colonized with either the R or HypoR community at 9, 15, 27, and 36 days post gavage.

FIG. 3D shows BugFACS analysis of feces obtained from R-colonized mice.

FIG. 4A is the experimental design of the co-housing study (Study 3). Four groups of 12 mice were dually-caged and fed the M18 diet. On day 3, two groups were colonized with the HypoR microbiota and two with the R microbiota. On day 10, one group harboring each microbiota was switched to the nutraceutical-supplemented M18 diet. On day 14, half the mice from each group were moved to a new, empty isolator, where they were each co-housed with another mouse as indicated.

FIG. 4B shows that mice fed the supplemented M18 diet exhibited increased CT-IgA ratios in their feces, and increased percentages of CD38loGL7+ cells among CD19+TCR-b− cells relative to mice fed the M18 diet. *, P<0.05, **, P<0.01, ***, P<0.001.

FIG. 4C is a heatmap showing the mean percent abundances of ASVs at 10, 13, 17, 28, and 40 days post-gavage in mice exposed to only the R microbiota, only the HypoR microbiota, or (through co-housing) the HypoR microbiota followed by the R microbiota (HypoR^Ch-R), or the R microbiota followed by HypoR microbiota (R^Ch-HypoR) in both the unsupplemented and nutraceutical supplemented M18 diet contexts. The column “Assigned microbiota” identifies ASVs previously demonstrated to be significantly associated with either the R or HypoR microbiota by indicator species analysis. The “Invade HypoR” and “Invade R” columns identify ASVs defined as invaders in the R->HypoR and HypoR->R directions, respectively. Only ASVs with a mean relative abundance of at least 1% in one mouse group on any day are included in the heatmap.

FIG. 4D Mice exposed to the R microbiota exhibited higher cecal concentrations of propionate than mice only exposed to HypoR microbiota. *, P<0.05,**, P<0.01, ***, P<0.001.

FIG. 4E Mice exposed to the R microbiota exhibited higher cecal concentrations of butyrate than mice only exposed to HypoR microbiota. *, P<0.05,**, P<0.01, ***, P<0.001.

FIG. 4F Mice exposed to the R microbiota exhibited higher cecal concentrations of succinate than mice only exposed to HypoR microbiota. *, P<0.05,***, P<0.001.

FIG. 5 shows the design of Study 4 (probiotic gavage), designed to study the effects of introducing (i) the intact R community, (ii) a 5-member consortium of cultured bacterial strains from the R community (Bacteroides acidifaciens, Bacteroides fragilis, Clostridioides difficile, Clostridium innocuum, Fusobacterium mortiferum), or (iii) Lactobacillus Rhamnosum GG (LGG) into the HypoR community.

FIG. 6 shows fecal anti-CT IgA/Total IgA data 7 d after the last CT vaccination from mice in Study 4 that were first colonized with the HypoR community and then gavaged with either R microbiota, the cultured 5 member R consortium or the Lactobacillus rhamnosus GG strain (LGG).

FIG. 7A shows the levels of activated memory B cells in the mesenteric lymph nodes in mice in Study 4.

FIG. 7B shows the levels of activated memory B cells in the spleens of mice in Study 4.

FIG. 8 shows the results 16S rDNA analysis of fecal samples collected from mice at the end of Study 4 showing the bacterial strains from the 5 member R cultured consortium (5memRCC) that were able to efficiently colonize mice previously gavaged with the HypoR microbiota.

FIG. 9 shows a heatmap of the bacterial taxa (Amplicon Sequence Variants; ASVs) that successfully invaded either the HypoR or R communities in the Study 3 (co-housing) and Study 4 (probiotic/direct gavage) experiments.

FIG. 10 shows ratios of fecal CT-specific IgA to total IgA in mice that were first colonized with the HypoR community and then gavaged with either the intact R microbiota or the cultured 5 member R culture consortium (5memRCC). *, P<0.05, **, P<0.01.

FIG. 11A,B show that HypoR mice exposed to the intact R microbiota or the 5 member R culture consortium (5memRCC) have higher concentrations of butyrate and propionate in their cecal contents than HypoR controls. *, P<0.05, ***, P<0.001.

FIG. 12A-C show features in the cecal metabolome identified by untargeted LC-Q-TOF MS that are associated with vaccine responsiveness in mice colonized with either the intact R community or the cultured 5 member R culture consortium (5memRCC). (A) m/z 144.1002 identified by collision-induced dissociation (CID) as proline betaine. (B) m/z 235.1078 and (C) its CID mass spectrum.

FIG. 13A-D show the results of non-targeted LC-QTOF MS of cecal contents obtained from HypoR mice subsequently colonized with the intact R community or the 5memRCC. (A) Principal components analysis of nontargeted metabolomic profiles separates the HypoR, HypoR+5memRCC and HypoR+R treatment groups. (B) Heatmap showing the concentrations of the top 10% of analytes with the strongest significant positive correlations with the CT-IgA ratio. (C) Heatmap showing the concentrations of analytes that differ between R^Ch-R, R^Ch-HypoRor HypoR^Ch-Rmice fed the M18 diet or the nutraceutical-supplemented M18 diet in the cohousing experiment described in Study 3. (D) Heatmap showing the concentrations of analytes in the supernatants of in vitro cultures of the 5memRCC and its individual members, as well as uninoculated controls.

FIG. 14A and FIG. 14B show the effect of lipid-coated encapsulation on the color intensity of spirulina.

FIG. 15 Molecular mimicry concept. Microbiota-derived crossreactive antigens may act to prime T cells and/or may act to prime B cells.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to a nutraceutical composition capable of increasing an immune response to an antigen or antigenic composition (i.e. a vaccine) in a mammal, the nutraceutical composition comprising a probiotic, spirulina, a cereal and a micronutrient. One embodiment provides a nutraceutical composition for improving a person's health by addressing malnutrition and boosting the immune system and improving vaccine effectiveness in vulnerable populations in Least Developed Countries (LDCs), including undernourished children, lactating and pregnant mothers, and the elderly population suffering from immunosenescence and the immunocompromised patients in industrialized countries. One embodiment provides a nutraceutical formulation that comprises a combined prebiotic and probiotic (together a synbiotic) formulation, wherein the nutraceutial formulation, when administered to a mammal, improves immune response in a mammal to an antigen, e.g. a vaccine.

An embodiment provides a nutraceutical composition comprising microbiota capable of conferring an augmented CT-IgA response to an antigen or antigenic composition (i.e. a vaccine) in a mammal and further comprises spirulina, amaranth, flaxseed and micronutrients.

In an embodiment of the invention there is provided a nutraceutical composition comprising a probiotic species selected from Bacillus sp., Bifidobacterium sp., Enterococcus sp., Lactobacillus sp., Lactococcus sp., Propionibacterium sp., Pediococcus sp., Saccharomyces sp. and Streptococcus spa Bacteroides, a Clostridioides, a Clostridium, an Erysipelotrichaceae, a Firmicutes, a Flavonifractor, a Fusobacterium, a Lactobacillus, a Parabacteroides, a Peptoclostridrium, a Robinsoniella, or a Subdoligranulum species; spirulina, a cereal and micronutrients.

One embodiment provides a nutraceutical composition comprising a probiotic species selected from a Bacteroides acidifaciens species, a Bacteroides fragilis species, a Clostridioides difficile species, a Clostridium innocuum species and a Fusobacterium mortiferum species; spirulina, a cereal and micronutrients. In certain embodiments, the microbiota C. difficile species does not possess genes encoding two principal glucosylating exotoxins, TcdA and TcdB.

Immune response to vaccines and natural antigens is often less robust in malnourished and vulnerable populations compared to well-nourished and healthy populations (Madhi, N Eng J Med 2010; Vesikary, Lancet 2007 and Bhandari, Lancet 2014). Thus, there is a need to develop a nutrient supplement/nutraceutical composition comprising microencapsulated prebiotic and/or probiotic (together being a ‘synbiotic’) wherein encapsulation may improve stability, encapsulated spirulina, cereals, and micronutrients comprising vitamins and minerals, such that the composition i) enhances or boosts the immune system and response to vaccines and/or natural antigens, particularly during the first 1000 days of an undernourished child's life; and ii) enhances or boosts the immune development and response to vaccines and/or natural antigens in vulnerable populations such as immunocompromised humans, pregnant females, lactating females and elderly humans.

Spirulina, which in its natural state is very green, also has an odor and a distinct taste. It is less palatable for humans in this form, so there is a need for encapsulated spirulina in a nutrient formulation capable of masking the undesirable flavour, odor and bright green color in order to obtain a galenic formulation more adapted for pediatric population. Therefore there is a need to provide encapsulated or coated spirulina and probiotic for use in a nutraceutical composition such that encapsulation does not negatively impact the nutritional value of the spirulina and probiotic present in the nutraceutial composition. In an embodiment of the invention, the spirulina present in the nutraceutical composition is spray-dried with a lipid formulation to coat or encapsulate the spirulina.

In embodiments of the invention, the coating or encapsulation materials are generally recognized as safe (GRAS), i.e. ingredients that can be used in food applications. In an embodiment, the lipids used to coat the spirulina are accepted as food additives, have a melting temperature higher than 40° C., and optionally have immunostimulating properties. In certain embodiments, the spray-dried spirulina coating comprises beta glucan, Microencapsulation materials may be applied using different microencapsulation techniques as described (Sultana, G. et al., “Encapsulation of probiotic bacteria with alginate-starch and evaluation of survival in simulated gastrointestinal conditions and in yoghurt” Int J Food Microbiol, 62 (2000), pp. 47-55; Muthukumarasamy et al., “Stability of Lactobacillus reuteri in different types of microcapsules” J Food Sci, 71 (2006), pp. 20-24; Anal and Singh “Recent advances in microencapsulation of probiotics for industrial applications and targeted delivery” Trends Food Sci Technol, 18 (2007), pp. 240-251; Ding and Shah “Effect of various encapsulating materials on the stability of probiotic bacteria” J Food Sci, 74 (2009), pp. M100-M107; Mokarram et al. “The influence of multistage alginate coating on survivability of potential probiotic bacteria in simulated gastric and intestinal juice” Food Res Int, 42 (2009), pp. 1040-1045; Chavarri et al. 2010; Cook et al. 2011, 2013). Spirulina may be coated by spray-drying with lipids to form a lipid-coated spray-dried spirulina. In certain embodiments, the spray-dried lipid-coated spirulina comprises a decreased color and or odor. In an embodiment, the spirulina may be spray-dried with plurol Oleique 497. In certain embodiments, the color of spirulina may be controlled through a higher lipid concentration, which decreases the green color of spirulina. In certain embodiments, the lipid used to prepare spray-dried spirulina comprises di- and tri-glyceride esters of fatty acids having a melting temperature of 40° C. or above. In certain embodiments, the lipid comprises Compritol 888 ATO (glyceryl dibehenate). In certain embodiments, the lipid comprises Gelucire 43/01. In certain In certain embodiments, the compritol 888 ATO has a melting range of 65-77° C. In certain embodiments, the Gelucire 43/01 has a melting temperature of 42° C.-46° C. In certain embodiments, the spray-dried spirulina coating comprises beta glucan, In certain embodiments, the spray-dried spirulina comprises ratio of 50% spirulina/50% lipid was kept for a better color and taste masking of spirulina.

In addition, certain embodiments provide the use of encapsulation of probiotic bacteria in milk proteins such as casein (Oliveira et al. “Stability of microencapsulated B. lactis (BI 01) and L. acidophilus (LAC 4) by complex coacervation followed by spray drying” J Microencapsul, 24 (2007), pp. 673-681; Heidebach et al. a “Microencapsulation of probiotic cells by means of rennet-gelation of milk proteins” Food Hydrocoll, 23 (2009), pp. 1670-1677 and Heidebach et al. “Transglutaminase-induced caseinate gelation for the microencapsulation of probiotic cells” Int Dairy J, 19 (2009), pp. 77-84), and whey protein (Doherty et al. “Survival of entrapped Lactobacillus rhamnosus GG in whey protein micro-beads during simulated ex vivo gastro-intestinal transit” Int Dairy J, 22 (2012), pp. 31-43).

Certain embodiments provide microencapsulation of probiotic bacteria in a natural polymer. Certain embodiments provide microencapsulation of probiotic species in alginate. Certain embodiments provide microencapsulation of probiotic bacteria in a pH-sensitive material (see Allan-Wojtas et al. “Microstructural studies of probiotic bacteria-loaded alginate microcapsules using standard electron microscopy techniques and anhydrous fixation” LWT Food Sci Technol, 41 (2008), pp. 101-108).

To fulfill many demands of a successful probiotic bacteria microencapsulation, different polymers may be used, natural and synthetic, to increase the resistance of such probiotic species against gastric conditions.

One embodiment of the invention provides a method of enhancing an immune response to a vaccine or antigen in a human, the method comprising administering an immune enhancing effective amount of a nutraceutical composition comprising: a probiotic species selected from Lactobacillus rhamnosus, Lactobacillus acidophilus; spirulina; cereal comprising flaxseed and amaranth; and micronutrients comprising i) vitamins B3, B6, C, D3, E and B9 and ii) minerals comprising magnesium, selenium and zinc; such that an enhanced immune response to a vaccine or antigen is observed in the human, as measured by an increase in mucosal vaccine specific IgA titer or as measured by an increase in systemic IgG titer.

One embodiment of the invention provides a method of using a probiotic to enhance the immune system in a human, the method comprising administering an immune enhancing effective amount of a probiotic species, wherein the probiotic species is a Bacteroides species, a Clostridium species, an Erysipelotrichaceae species, a Firmicutes species, a Flavonifractor species, a Fusobacterium species, a Lactobacillus species, a Parabacteroides species, a Peptoclostridrium species, a Robinsoniella species, a Subdoligranulum species or a combination thereof, such that the enhanced immune system is observable by an increased response to a vaccine or antigen in the human, as measured by an increase in mucosal vaccine specific IgA titer or as measured by an increase in systemic IgG titer.

One embodiment of the invention provides use of a nutraceutical composition as described herein for enhancing the immune system in a human by increasing an immune response to an antigen or a vaccine in said human, as measured by an increase in mucosal vaccine specific IgA titer or as measured by an increase in systemic IgG titer.

Embodiments of the invention relate to methods for improving an immune response and/or an IgA (CT-IgA) response in a mammal to an antigen or an antigenic composition (i.e. a vaccine), the method comprising administering a nutraceutical composition capable of increasing an immune response in the mammal, wherein the nutraceutical composition comprises a prebiotic, a probiotic or a synbiotic; spirulina; a cereal; and a micronutrient, such that an improved immune response and/or increase in vaccine specific IgA is observed.

One embodiment provides a method for increasing an immune response to an antigen or an antigenic composition such as a vaccine in a mammal, wherein the increased immune response may include an increase in germinal center memory B-cells in mesenteric lymph nodes, wherein the method comprising administering a nutraceutical composition capable of increasing an immune response as described herein to the mammal, wherein the increased immune response and/or increase in germinal center memory B-cells may be accompanied by changes in the cecal metabolome, including elevated short chain fatty acids.

One embodiment provides a method for increasing an immune response to an antigenic composition (e.g. an oral cholera toxin vaccine) wherein the method comprises administering to the mammal a nutraceutical composition capable of conferring augmented CT-IgA responses in the mammal.

One embodiment provides a nutraceutical composition comprising a prebiotic, spirulina and micronutrients. In particular embodiments, the prebiotic comprises a cereal, more specifically the prebiotic comprises flaxseed, amaranth, rice, oats, teff, bran, barley, wheat, rye, maize, millet, buckwheat, spelt, chia, quinoa or any other grain. In particular embodiments, the micronutrients comprise a vitamin and mineral. Particular embodiments provide a nutraceutical composition comprising a prebiotic, spirulina, micronutrients and a probiotic species. In particular embodiments, the probiotic comprises a Bacteroides species, a Fusobacterium species, a Clostridioides species, a Clostridium species, or a combination thereof. In particular embodiments, the probiotic comprises a Bacteroides acidifaciens species, a Bacteroides fragilis species, a non-toxigenic Clostridioides difficile species, a Clostridium innocuum species, a Fusobacterium mortiferum species or a combination thereof. In particular embodiments, the probiotic species is Fusobacterium mortiferum strain 9G6, Bacteroides acidifaciens strain 9G3, Bacteroides fragilis strain 8E3, Clostridium innocuum strain 9H7, Clostridioides difficile strain 9C4 or a combination thereof. In particular embodiments, the probiotic comprises a Bacillus sp., Bifidobacterium sp., Enterococcus sp., Lactobacillus sp., Lactococcus sp., Propionibacterium sp., Pediococcus sp., Saccharomyces sp. and Streptococcus spa Bacteroides, a Clostridioides, a Clostridium, an Erysipelotrichaceae, a Firmicutes, a Flavonifractor, a Fusobacterium, a Lactobacillus, a Parabacteroides, a Peptoclostridrium, a Robinsoniella, a Subdoligranulum species, or a combination thereof.

In particular embodiments, there is provided a nutraceutical composition comprising a probiotic; spirulina; a cereal; and micronutrients, wherein the probiotic comprises a probiotic selected from a Bacteroides species, a Clostridioides species, a Clostridium species, an Erysipelotrichaceae species, a Firmicutes species, a Flavonifractor species, a Fusobacterium species, a Lactobacillus species, a Parabacteroides species, a Peptoclostridrium species, a Robinsoniella species, a Subdoligranulum species, or a combination thereof. In particular embodiments, the probiotic comprises a Bacillus sp., Bifidobacterium sp., Enterococcus sp., Lactobacillus sp., Lactococcus sp., Propionibacterium sp., Pediococcus sp., Saccharomyces sp., a Streptococcus sp., or a combination thereof. In particular embodiments the probiotic comprises a Bacteroides species, a Clostridioides species, a Clostridium species, an Erysipelotrichaceae species, a Firmicutes species, a Flavonifractor species, a Fusobacterium species, a Lactobacillus species, a Parabacteroides species, a Peptoclostridrium species, a Robinsoniella species, a Subdoligranulum species or a combination thereof. In particular embodiments, the probiotic species comprises a Bacteroides species, a Fusobacterium species, a Clostridioides species, a Clostridium species, or a combination thereof. In particular embodiments, the probiotic species comprises a Bacteroides acidifaciens species, a Bacteroides fragilis species, a non-toxigenic Clostridioides difficile species, a Clostridium innocuum species, a Fusobacterium mortiferum species or a combination thereof. In particular embodiments, the probiotic species is Fusobacterium mortiferum strain 9G6, Bacteroides acidifaciens strain 9G3, Bacteroides fragilis strain 8E3, Clostridium innocuum strain 9H7, Clostridioides difficile strain 9C4 or a combination thereof.

Certain embodiments described herein provide a nutraceutical composition comprising a probiotic comprises a Bacillus sp., a Bifidobacterium sp., a Enterococcus sp., a Lactobacillus sp., a Lactococcus sp., a Propionibacterium sp., a Pediococcus sp., Saccharomyces sp., and a Streptococcus sp., Particular embodiments provide a nutraceutical composition wherein the probiotic comprises a Bacteroides species, a Clostridioides species, a Clostridium species, an Erysipelotrichaceae species, a Firmicutes species, a Flavonifractor species, a Fusobacterium species, a Lactobacillus species, a Parabacteroides, a Peptoclostridrium, a Robinsoniella, or a Subdoligranulum species. Particular embodiments provide a nutraceutical composition comprising a probiotic, wherein the probiotic comprises a Bacteroides species, a Clostridium species, an Erysipelotrichaceae species, a Firmicutes species, a Flavonifractor species, a Fusobacterium species, a Lactobacillus species, a Parabacteroides, a Clostridioides species; a Peptoclostridrium, a Robinsoniella, or a Subdoligranulum species. In particular embodiments, the probiotic comprises a Bacteroides species, a Fusobacterium species, a Parabacteroides, species a Clostridioides species, or a Clostridium species or a combination thereof. In particular embodiments, the probiotic comprises a Bacteroides acidifaciens species, a Bacteroides fragilis species, a non-toxigenic Clostridioides difficile species, a Clostridium innocuum species, Fusobacterium mortiferum species or a combination thereof. In particular embodiments, the probiotic comprises Fusobacterium mortiferum strain 9G6, Bacteroides acidifaciens strain 9G3, Bacteroides fragilis strain 8E3, Clostridium innocuum strain 9H7, Clostridioides difficile strain 9C4 or a combination thereof.

In a particular embodiment, the nutraceutical composition comprises a composition as described herein, comprising a non-toxigenic Clostridioides species. In particular embodiments, the non-toxigenic Clostridioides difficile species does not possess genes encoding glucosylating exotoxins TcdA and TcdB.

In a particular embodiment, a nutraceutical composition as described herein comprises a cereal, wherein the cereal comprises flaxseed, amaranth, rice, oats, teff, bran, barley, wheat, rye, maize, millet, buckwheat, spelt, chia, quinoa or any other grain. In a particular embodiment, a nutraceutical composition as described herein comprises a cereal, wherein the cereal comprises flaxseed and amaranth. In a particular embodiment, a nutraceutical composition as described herein comprises micronutrients, wherein the micronutrients comprise a vitamin and mineral. In a particular embodiment of a nutraceutical composition that comprises micronutrients as described herein, the micronutrients comprise i) a vitamin A (α-carotene, β-carotene, retinol), a vitamin B1 (thiamin), a vitamin B2 (riboflavin), a vitamin B3 (niacin), a vitamin B5 (pantothenic acid), a vitamin B6 (pyroxidine), a vitamin B7 (biotin), a vitamin B9 (folate or folic acid), a vitamin B12 (cobalamin or cyanocobalamin), a vitamin C (ascorbic acid or ascorbate), a vitamin D1 (a mixture of lumisterol and califerol), a vitamin D2 (ergocalciferol), a vitamin D3 (cholecalciferol), a vitamin E (α-tocopherol) or a vitamin K (phytonadione), and ii) a mineral comprising calcium, chloride, chromium, copper, iodine, iron, magnesium, manganese, molybdenum, phosphorus, potassium, selenium, sodium or zinc. In a particular embodiment of a nutraceutical composition that comprises micronutrients as described herein, the micronutrients comprise vitamin B3, vitamin B6, vitamin C, vitamin D3, vitamin E or vitamin B9. In a particular embodiment of a nutraceutical composition that comprises micronutrients as described herein, the micronutrients comprise magnesium, selenium or zinc.

In a particular embodiment, a nutraceutical composition as described herein comprises a spirulina, wherein the spirulina is encapsulated. In particular embodiments, the encapsulated spirulina is encapsulated with a lipid carrier emulsion. In particular embodiments, the lipid carrier emulsion comprises a nonionic emulsifier. In particular embodiments, the nonionic emulsifier comprises an oleic acid, a dibehenate, a di- and triglyceride ester of a fatty acid, a beta-glucan, or a combination thereof. In particular embodiments, the lipid carrier emulsion comprises a microemulsion. In particular embodiments, the lipid carrier microemulsion comprises an oleic acid. In particular embodiments, the lipid carrier microemulsion comprises a dibehenate. In particular embodiments, the lipid carrier microemulsion comprises a di- or tri-glyceride. In particular embodiments, the lipid carrier microemulsion comprises a beta-glucan. In particular embodiments, the lipid carrier microemulsion comprises Plurol® Oleique CC 497 CG, Compritol 888 ATO, Gelucire 43/01.

In a particular embodiment as described herein, the lipid carrier microemulsion is formulated with the spirulina at a concentration of up to 1%, 5%, 10% 15%, 20%, 30% 40% or 50% (by weight).

In a particular embodiment, when a nutraceutical as described herein comprises a spirulina, a cereal, and micronutrients, the spirulina is present in an amount of 5-15% (dry weight), the cereal comprises flaxseed and amaranth, and the flaxseed is present in an amount of 1-5% (dry weight), the amaranth is present in an amount of 5-15% (dry weight) and the micronutrients are present in an amount of 0.02-0.05% (dry weight). In a particular embodiment, the spirulina is present in an amount of 5% (dry weight), the flaxseed is present in an amount of 2.5% (dry weight), the amaranth is present in an amount of 10% (dry weight) and the micronutrients are present in an amount of 0.025% (dry weight).

In a particular embodiment, a nutraceutical composition as described herein comprises a probiotic, wherein the probiotic is encapsulated. In a particular embodiment, the encapsulated probiotic is microencapsulated. In a particular embodiment, a nutraceutical composition as described herein comprises a microbiota metabolite, including a metabolite as set forth in any of FIG. 12A, FIG. 12B, 12C and/or FIG. 13C, or a combination thereof. In a particular embodiment, a nutraceutical composition as described herein comprises an an adjuvant. In a particular embodiment, the adjuvant is microbiota metabolite. In a particular embodiment, the microbiota metabolite comprises a metabolite as set forth in any of FIG. 12A, FIG. 12B, 12C and/or FIG. 13C, or a combination thereof.

One embodiment of the invention provides a method of enhancing an immune response to a vaccine or antigen in a human, the method comprising administering an immune enhancing effective amount of a nutraceutical composition as described herein. In a particular embodiment, the method of enhancing an immune response to a vaccine or antigen in a human comprises administering a nutraceutical composition as described herein comprising a probiotic. In particular embodiments, the probiotic comprises a Lactobacillus rhamnosus species, Lactobacillus acidophilus species;

In a particular embodiment, the method of enhancing an immune response to a vaccine or antigen in a human comprises administering a nutraceutical composition as described herein comprising a spirulina. In a particular embodiment, the method of enhancing an immune response to a vaccine or antigen in a human comprises administering a nutraceutical composition as described herein comprising a cereal. In particular embodiments, the cereal comprises flaxseed and amaranth. In a particular embodiment, the method of enhancing an immune response to a vaccine or antigen in a human comprises administering a nutraceutical composition as described herein comprising micronutrients and minerals. In particular embodiments, the micronutrients comprises vitamin B3, vitamin B6, vitamin C, vitamin D3, vitamin E and vitamin B9; and the minerals comprise magnesium, selenium and zinc. In a particular embodiment, the method of enhancing an immune response to a vaccine or antigen in a human comprises administering a nutraceutical composition as described herein such that an enhanced immune response to a vaccine or antigen is observed in the human, as measured by an increase in mucosal IgA titer or as measured by an increase in systemic IgG titer. In particular embodiments, the human is selected from a child, a female, an elderly human, an immunocompromised human or an immunosenescent human. In particular embodiments, the human is a child and more particularly, the child is an undernourished child. In particular embodiments, the human is a female, more particularly the female is a pregnant female. In a particular embodiment, the female is a lactating female. In particular embodiments, the human is an elderly human. In particular embodiments, the human is an immunocompromised human, more particularly, the immunocompromised human has been diagnosed with cancer, or is infected with a virus including human immunodeficiency virus (HIV), hepatitis B virus (HBV), hepatitis D virus (HDV), hepatitis C virus (HCV), Dengue virus, Influenza virus, Zika virus, or Epstein-barr virus.

In a particular embodiment of a nutraceutical composition as described herein, the nutraceutical composition is formulated as a powder, a tablet, a capsule, a bead, a gel, a paste, or a liquid. In a particular embodiment, the nutraceutical composition is formulated as a powder that is reconstituted in liquid, more particularly, the powder is reconstituted in up to 50, 75, 100, 150, 200, 250, 300 or 400 mL of water.

In a particular embodiment of a nutraceutical composition as described herein, the nutraceutical composition is provided at a dose of up to 10, 20, 30, 40, 50 or 100 g/kg body weight, more preferably at a dose of up to 0.5, 1, 2, 3, 4, 5 or 10 g/kg body weight. In a particular embodiment of a nutraceutical composition as described herein the nutraceutical composition is administered orally.

In a particular embodiment of a nutraceutical composition as described herein, the nutraceutical composition is administered 1× to 3× daily, or is administered 1× to 6× weekly, or is administered biweekly, or is administered monthly. In a particular embodiment of a nutraceutical composition as described herein, the nutraceutical composition is administered 1, 2, 3, 4, 5 or 6 months prior to an immunization. In a particular embodiment of a nutraceutical as described herein, the nutraceutical composition is administered 1, 2, 3 4, 5 or 6 months post-immunization. In a particular embodiment of a nutraceutical as described herein, the nutraceutical composition is further administered 1, 2, 5, 10, 20 or 30 years post-immunization. In a particular embodiment of a nutraceutical as described herein, the nutraceutical composition is administered over the lifetime of the human.

In a particular embodiment there is provided a method of enhancing an immune response to a vaccine or antigen in a human, the method comprising administering a nutraceutical composition as described herein, wherein the nutraceutical composition further comprises a metabolite as set forth in any of FIG. 12A, FIG. 12B, 12C and/or FIG. 13C, or a combination thereof.

In a particular embodiment there is provided a method of enhancing an immune response to a vaccine or antigen in a human, the method comprising administering an immune enhancing effective amount of a probiotic, wherein the probiotic comprises a Bacteroides species, a Clostridioides species, Clostridium species, an Erysipelotrichaceae species, a Firmicutes species, a Flavonifractor species, a Fusobacterium species, a Lactobacillus species, a Parabacteroides species, a Peptoclostridrium species, a Robinsoniella species, a Subdoligranulum species or a combination thereof, such that the enhanced immune system is observable by an increased response to a vaccine or antigen in the human, as measured by an increase in mucosal IgA titer or as measured by an increase in systemic IgG titer. In a particular embodiment, the method comprises administering an immune enhancing effective amount of a probiotic, wherein the probiotic comprises a Fusobacterium species, a Clostridioides species, a Clostridium species, or a combination thereof. In a particular embodiment, the method comprises administering an immune enhancing effective amount of a probiotic, wherein the probiotic comprises a Bacteroides acidifaciens species, a Bacteroides fragilis species, a non-toxigenic Clostridioides difficile species, a Clostridium innocuum species, a Fusobacterium mortiferum species or a combination thereof. In a particular embodiment, the method comprises administering an immune enhancing effective amount of a probiotic, wherein the probiotic comprises Fusobacterium mortiferum strain 9G6, Bacteroides acidifaciens strain 9G3, Bacteroides fragilis strain 8E3, Clostridium innocuum strain 9H7, Clostridioides difficile strain 9C4 or a combination thereof.

In a particular embodiment wherein there is provided a method of enhancing an immune response to a vaccine or antigen in a human, wherein the method comprises administering an immune enhancing effective amount of a probiotic, wherein the human is a child, a female, or an elderly human. In a particular embodiment there is provided a method of enhancing an immune response to a vaccine or antigen in a human, wherein the human is a child. In a particular embodiment, the child is an undernourished child. In a particular embodiment wherein there is provided a method of enhancing an immune response to a vaccine or antigen in a human, wherein the human is a female. In a particular embodiment, the female is a pregnant female. In a particular embodiment, the female is a lactating female. In a particular embodiment wherein there is provided a method of enhancing an immune response to a vaccine or antigen in a human, the human is an elderly human. In a particular embodiment, the human is an immunocompromised human. More particularly, the immunocompromised human has been diagnosed with cancer, or is infected with a virus including human immunodeficiency virus (HIV), hepatitis B virus (HBV), hepatitis D virus (HDV), hepatitis C virus (HCV), Dengue virus, Influenza virus, Zika virus, or Epstein-barr virus.

One particular embodiment provides use of a nutraceutical composition as described herein for enhancing the immune system by increasing an immune response to an antigen or a vaccine in a human, as measured by an increase in mucosal IgA titer or as measured by an increase in systemic IgG titer. In a particular embodiment there is provided use of a nutraceutical composition as described herein for enhancing the immune system by increasing an immune response to an antigen or a vaccine in a human, wherein the human is a child, a female, or an elderly human. In a particular embodiment of use of a nutraceutical composition as described herein for enhancing the immune system by increasing an immune response to an antigen or a vaccine in a human, the human is a child, and more particularly the child is an undernourished child. In a particular embodiment of use of a nutraceutical composition as described herein for enhancing the immune system by increasing an immune response to an antigen or a vaccine in a human, the human is a female. In particular embodiments, the female is a pregnant female. In particular embodiments, the female is a lactating female. In a particular embodiment of use of a nutraceutical composition as described herein for enhancing the immune system by increasing an immune response to an antigen or a vaccine in a human, the human is an elderly human. In a particular embodiment of use of a nutraceutical composition as described herein for enhancing the immune system by increasing an immune response to an antigen or a vaccine in a human, the human is an immunocompromised human; more particularly, the immunocompromised human has been diagnosed with cancer, or is infected with a virus including human immunodeficiency virus (HIV), hepatitis B virus (HBV), hepatitis D virus (HDV), hepatitis C virus (HCV), Dengue virus, Influenza virus, Zika virus, or Epstein-barr virus.

One embodiment of the invention provides use of a nutraceutical composition as described herein in the manufacture of a medicament for the enhancement of an immune response in a human. In a particular embodiment of a use of a nutraceutical composition as described herein in the manufacture of a medicament for the enhancement of an immune response in a human, the human is a child, a female, or an elderly human.

In a particular embodiment of a use of a nutraceutical composition as described herein in the manufacture of a medicament for the enhancement of an immune response in a human, the human is a child. In a particular embodiment, the child is an undernourished child. In a particular embodiment of a use of a nutraceutical composition as described herein in the manufacture of a medicament for the enhancement of an immune response in a human, the human is a female. In a particular embodiment, the female is a pregnant female. In a particular embodiment, the female is a lactating female. In a particular embodiment of a use of a nutraceutical composition as described herein in the manufacture of a medicament for the enhancement of an immune response in a human, the human is an elderly human. In a particular embodiment of a use of a nutraceutical composition as described herein in the manufacture of a medicament for the enhancement of an immune response in a human, the human is an immunocompromised human. In a particular embodiment, the immunocompromised human has been diagnosed with cancer, or is infected with a virus including human immunodeficiency virus (HIV), hepatitis B virus (HBV), hepatitis D virus (HDV), hepatitis C virus (HCV), Dengue virus, Influenza virus, Zika virus, or Epstein-barr virus.

Definitions

“5memRCC” refers to the 5 member bacterial consortium cultured from a vaccine responsive (R) microbiota.

“ANOVA” as used herein stands for Analysis Of VAriance, a statistical method that divides variation in a set of observations into distinct components. It can be used to determine whether there are any statistically significant differences between the means of three of more independent (unrelated) groups. For example, the one-way ANOVA tests the null hypothesis:

$H_{0} : μ_{1} = μ_{2} = \dots = μ_{κ}$

wherein μ=group mean and κ=number of groups, and if this analysis provides a statistically significant result, it indicates that at least two group means are statistically different from each other.

“Anthropometric” as used herein means measurements or scientific study of measurements and proportions of the human body. Examples include height, weight, body mass index, mid-upper arm circumference (MUAC), triceps skin fold (TSF) circumference, mid-are muscle circumference, head circumference and the like.

“ASV” as used herein means amplicon sequence variant.

“Base diet” as used herein means the Mirpur-18 (M18) diet set forth in Table 2A, or any of the base diet variations described herein, by component and/or by weight, of the M18 diet of Table 2A.

“Cereal” as used herein means the edible component or grain portion of any grass.

CT=Cholera Toxin (CT)

CTB=the B subunit of cholera toxin

d=day or days

“Encapsulation” or “encapsulated” as used herein means to coat, encompass, surround, associate or adhere one substance around or to another substance being encapsulated. Substances that may be encapsulated include the probiotics, spirulina or any other component of the nutraceutical compositions describe herein.

“F4V” as used herein means nutraceutical compositions/supplements. F4V, as used in the present application and figures, and is synonymous with Probiotic (−) Nutraceutical Composition I, or just Nutraceutical Composition I.

“HAZ” as used herein refers to World Health Organization (WHO) recommended use of height-for-age Z scores (WHZ) to assess malnutrition prevalence in children.

“Mirpur-18 base diet”, first described by Gehrig et al. “Effects of microbiota-directed foods in gnobiotic animals and undernourished children”, Science 365, 6449 and referred to herein, is also referred to as Mirpur-18 diet or M18 diet or M18, and is described herein in Table 2A.

“Supplemented Mirpur-18 diet”, also referred to supplemented M18 diet, supplemented M18 or Nutraceutical Composition I-supplemented M18 diet is described herein in Table 2B.

“An immune enhancing effective amount of a nutraceutical composition” as used herein means an amount of nutraceutical composition sufficient to provoke an enhanced immune response in a subject, including a human, to an antigen or to a vaccine, as measured by an increase in mucosal vaccine specific IgA titer or as measured by an increase in systemic IgG titer.

“LDC” or “LDCs” as used herein means Least Developed Countries. Least Developed Countries includes, but is not limited, to the UN list of Least Developed Countries (updated March 2018) which includes Afghanistan, Angola, Bangladesh, Benin, Bhutan, Burkina Faso, Burundi, Cambodia, Central African Republic, Chad, Camoros, Democratic Replublic of Congo, Djobouti, Eritrea, Ethiopia, Gambia, Guinea, Guinea-Bissau, Haiti, Kiribati, Lao People's Democratic Republic, Lesotho, Liberia, Madagascar, Malawi, Mali, Mauritania, Mozambique, Myanmar, Nepal, Niger, Rwanda, Sao Tome and Principe, Senegal, Sierra Leone, Solomon Islands, Somalia, South Sudan, Sudan, Timor-Leste, Togo, Tuvalu, Uganda, United Republic of Tanzania, Vanuatu, Yemen, Zambia (https://www.un.org/development/desa/dpad/wp-content/uploads/sites/45/publication/Idc_list.pdf).

“Microbiome” as used herein means all of the genetic material that makes up or is contained in the human gut microbiota.

“Microbiota” as used herein refers to the entire collection of microorganisms in the human gut.

“Microemulsion” as used herein means a system wherein one phase, the dispersed phase (such as an oil or lipid), is solubilized in another phase, the continuous phase (such as water), to form a new component known as an emulsifier. Microemulsions form spontaneously, typically with formation of small droplets (micelles of the dispersed phase within the continuous phase) with diameters of 10-100 nm. Examples of lipids to be used in a microemulsion is Plurol® Oleique CC 497 CG (polyglycerol ester of oleic acid—CAS 9007-48-1), which is available from Gattefossé (https://www.ulprospectorcom/en/eu/PersonalCare/Detail/3983/113082/Plurol-Oleique-CC-497-CG), Compritol 888 ATO and Gelucire 43/01 which are also available from Gattefossé, and beta-glucans, which act as immunomodulator agents that can boost the immune system by stimulating activity of macrophages and lymphocytes. Thus, coating spirulina with beta-glucans may result in synergies.

“Micronutrient(s)” as used herein refers to a substance, often present in trace amounts, important or useful for the normal growth and development of an animal, including a human. A micronutrient may be a vitamin, mineral or compound, such as an amino acid, co-enzyme, fatty acid, or a neurotransmitter precursor such as choline. A micronutrient may be an element or compound not produced by the animal which relies on the micronutrient for normal growth, such as calcium or iron or zinc. A micronutrient may also be a compound or substance that is produced only in limited amounts by the animal which relies on it for normal growth.

MLN=mesenteric lymph nodule(s)

“MLN Treg(s)” as used herein refers to T regulatory cell(s) in mesenteric lymph node(s)

mo=month or months

“Non-toxigenic” as used herein refers to a bacterial species that does not possess genes encoding endotoxins which may mediate pathogenic effects. Specifically, by example, a non-toxigenic Clostridioides difficile species will not possess genes encoding the glucosylating exotoxins TcdA and Tcd B.

“Nutraceutical” as used herein refers to an edible additive for food or beverage such that the substance provides a medicinal, health or immunological benefit to an animal, including a human, that ingests the nutraceutical. The term nutraceutical originally was coined as a combination of the words “nutrition” and “pharmaceutical”. A nutraceutical may be a food/beverage or food/beverage component, such as a dietary supplement or a food additive. As used herein, the term food is intended to encompass any edible substance, which substance may be in solid, liquid, paste, tablet or other orally ingestible form. A nutraceutical may also supplement the diet, and includes traditional dietary supplements such as vitamins, minerals, herbs, oils and substances such as glucosamine, amino acids and other dietary supplements. As used herein, a nutraceutical is intended for oral ingestion and provides a health, medicinal or immunological benefit that may aid in disease prevention, disease treatment, and immunologic response. A nutraceutical may be a pharmaceutical-grade nutrient with standardized properties. A nutraceutical may also be a combination of substances including any combination of dietary supplements, food additives, vitamins, minerals, probiotics, prebiotics, spirulina, cereals and other substances that confer a health benefit to the animal, including a human, that ingests the nutraceutical. A nutraceutical may also be considered a functional food or functional food ingredient that provides a health, medicinal or immunological benefit in addition to the basic nutritional value of the food or food ingredient. A nutraceutical may be any functional or medicinal food that plays a role in maintaining well being, enhancing health, modulating immunity and thereby aiding in preventing as well as treating specific diseases.

“OTU(s)” as used herein means operational taxonomic unit(s)

“PERMANOVA” as used herein refers to PERmutational Multivariate ANalysis Of VAriance

“Prebiotic” as used herein refers to any substance that when consumed or administered provides non-digestible fibers that, when passed through the intestine and reach the colon, are fermented by microflora in the gut. Prebiotic(s) include cereals, dietary fiber, carbohydrates, polysaccharides and oligosaccharides, peptides and peptidocarbohydrates and peptidosaccharides. Prebiotics are fermented by beneficial bacteria in the gut as a source of fuel and to enhance gut flora health. Prebiotics can be considered to feed probiotics and gut microflora.

“Probiotic” as used herein refers to any substance(s) that when consumed or administered stimulates the growth of microorganisms in an animal, including a human, especially those bacteria with beneficial properties. Probiotics include live micro-organisms that confer a health benefit on the host. Examples of probiotics include the intestinal microorganism flora (microflora) of an animal, including a human, certain foods such as cheese and yogurt which contain beneficial bacteria, and dietary supplements such as powdered probiotic drink formulas or powdered probiotics available in capsules. Probiotics are capable of maintaining and/or restoring beneficial bacteria to the digestive tract.

sp.=species

- “spirulina” as used herein means a filamentous cyanobacteria (microscopic blue-green algae) that form tangled masses in warm alkaline lakes in Africa and Central and South America. The two most commonly utilized species of spirulina are Arthrospira platensis and Arthrospira maxima.

“Synbiotic” as used herein means a combined prebiotic and probiotic.

TEER as used herein refers to Transepithelial Electrical Resistance.

“WAZ” as used herein refers to World Health Organization (WHO) recommended use of weight-for-age Z scores (WHZ) to assess malnutrition prevalence in children.

“WHZ” as used herein refers to World Health Organization (WHO) recommended use of weight-for-height Z scores (WHZ) to assess malnutrition prevalence in children.

Z score” as used herein refers to a malnutrition score using various measurements to arrive at the Z score. In statistics, a Z-score is also referred to as a standard score, and it provides an idea of how far from the mean a given data point is. A Z-score, when placed on a normal (standard) distribution curve will range from −3 standard deviations to +3 standard deviations from the mean. A Z-score can be determined if the mean (μ) and population standard deviation (σ) is known. The WHO Global Database on Child Growth and Malnutrition uses a Z-score cut-off of <−2 sd (standard deviation) from the mean weight-for-age, height-for-age and weight-for-height values in children to define low malnutrition; >−3 sd from mean weight-for-age, height-for-age and weight-for-height in children to define severe malnutrition; and >−2 sd and <−3 sd from weight-for-age, height-for-age and weight-for-height in children, to define moderate malnutrition in children.

Embodiments herein relating to “vaccine compositions” are also applicable to embodiments relating to “immunogenic compositions”, and vice versa, wherein each term may be used interchangeably to describe a composition which provides an immunostimulatory effect upon administration to an animal.

Nutraceutical Compositions/Routes of Administration/Dosages

Compositions—General

In embodiments of the invention, nutraceutical compositions or supplements suitable for boosting an immune response, for example as a nutritive oral adjuvant for vaccination, will generally comprise spirulina, cereals, micronutrients, and may comprise probiotics or no probiotic. Spirulina may be coated or encapsulated. In certain embodiments, encapsulation or coating may help to mask color, odor and/or flavour. In embodiments, cereals may be selected from flaxseed, amaranth, teff, rice, oats, bran, barley, wheat, rye, maize, millet, buckwheat, spelt, chia, quinoa or any other grain. In embodiments of the invention, micronutrients include vitamins and minerals. In embodiments, vitamins may be selected from vitamin A (α-carotene, β-carotene, retinol), B1 (thiamin), B2 (riboflavin), B3 (niacin), B5 (pantothenic acid), B6 (pyroxidine), B7 (biotin), B9 (folate or folic acid), B12 (cobalamin or cyanocobalamin), C (ascorbic acid or ascorbate), D1 (a mixture of lumisterol and califerol), D2 (ergocalciferol), D3 (cholecalciferol), E (α-tocopherol) and K (phytonadione). In embodiments of the invention, minerals may be selected from calcium, chloride, chromium, copper, iodine, iron, magnesium, manganese, molybdenum, phosphorus, potassium, selenium, sodium and zinc.

Nutraceutical compositions may also comprise fish oil such as cod-liver oil; rapeseed or rapeseed oil; lipid compounds such as fatty acids including omega-3 and omega-6 fatty acids; phospholipids; ceramides; and sterols including phytosterols, zoosterols and mycosterols. Examples of phytosterols include avenosterol, β-sitosterol, campesterol and stigmasterol, as well as fully-saturated phytosterols known as phytostanols such as sitostanol and coprostanol. Examples of zoosterols include cholesterol, 24-isopropylcholesterol, 7-dehydrocholesterol, lanosterol, nicasterol, oxysterol, 4-methylcholestan-8(14), 24-diene-3β-ol, ganoderic acid and gorgosterol. Examples of mycosterols include ergosterol, antrosterol and saringosterol.

Examples of probiotic that may be added to any nutraceutical composition in embodiments herein include a Bacillus sp., a Bifidobacterium sp., an Enterococcus sp., a Lactobacillus sp., a Lactococcus sp., a Pediococcus sp., a Saccharomyces sp., a Streptococcus sp., a Bacteroides sp., a Clostridioides sp., a Clostridium sp., an Erysipelotrichaceae sp., a Firmicutes sp., a Flavonifractor sp., a Fusobacterium sp., a Lactobacillus sp., a Parabacteroides sp., a Peptoclostridrium sp., a Robinsoniella sp., or a Subdoligranulum species.

Examples of specific species include Bacillus coagulans, Bacillus laterosporus, Bifidobacterium breve, Bifidobacterium bifidum, Bifidobacterium infantis, Bifidobacterium lactis, Bifidobacterium longum, Enterococcus faecium, Lactobacillus acidophilus, Lactobacillus brevis, Lactobacillus bulgaricus, Lactobacillus casei, Lactobacillus fermentum, Lactobacillus gasseri, Lactobacillus helveticus, Lactobacillus infantis, Lactobacillus lactis, Lactobacillus plantarum, Lactobacillus paracasei, Lactobacillus rhamnosus, Lactobacillus salivarius, Lactobacillus reuteri, Lactococcus lactis, Pediococcus acidilactici, Saccharomyces boulardii, Streptococcus thermophilus Bacteroides acidifaciens, Bacteroides fragilis, Clostridioides difficile, Clostridium innocuum and Fusobacterium mortiferum. In certain embodiments, the probiotic species is Fusobacterium mortiferum strain 9G6, Bacteroides acidifaciens strain 9G3, Bacteroides fragilis strain 8E3, Clostridium innocuum strain 9H7, or Clostridioides difficile strain 9C4. Other examples of probiotics that may be added in embodiments herein to any nutraceutical composition described herein include new probiotics experimentally identified as having adjuvant properties for increasing an immune response to an antigen, referred to herein as a “probiotic adjuvant”, whether the antigen is encountered naturally or via vaccination with an immunogenic composition, e.g. a vaccine. In embodiments, an experimentally identified probiotic adjuvant may be identified by comparing immune response to the antigen in the presence of other probiotics, and selecting those probiotics that impart improved immune response to an antigen compared to other probiotic and/or compared to the immune response observed to the antigen without the adjuvant probiotic.

In certain embodiments, a nutraceutical composition may comprise spirulina, cereals, micronutrients, and may comprise probiotics or no probiotic. In certain embodiments, the spirulina in the composition may be coated or encapsulated to, e.g., help mask color, odor and/or flavour of the spirulina. In certain embodiments, the cereals of the nutraceutical composition may comprise cereals selected from flaxseed, amaranth, teff, rice, oats, bran, barley, wheat, rye, maize, millet, buckwheat, spelt, chia, quinoa or any other grain. In certain embodiments, the micronutrients of the nutraceutical composition include vitamins and minerals wherein the vitamins are selected from vitamin A (α-carotene, β-carotene, retinol), B1 (thiamin), B2 (riboflavin), B3 (niacin), B5 (pantothenic acid), B6 (pyroxidine), B7 (biotin), B9 (folate or folic acid), B12 (cobalamin or cyanocobalamin), C (ascorbic acid or ascorbate), D1 (a mixture of lumisterol and califerol), D2 (ergocalciferol), D3 (cholecalciferol), E (α-tocopherol) and K (phytonadione), and wherein the minerals are selected from calcium, chloride, chromium, copper, iodine, iron, magnesium, manganese, molybdenum, phosphorus, potassium, selenium, sodium and zinc.

In certain embodiments the nutraceutical compositions as described herein may optionally comprise: fish oil such as cod-liver oil; rapeseed or rapeseed oil; a lipid compound such as a fatty acid including an omega-3 or an omega-6 fatty acid; a phospholipid; a ceramide; or a sterol including a phytosterol, zoosterol or mycosterol. In certain embodiments the phytosterol is selected from β-sitosterol, campesterol and stigmasterol. In certain embodiments, the zoosterol is cholesterol. In certain embodiments, the mycosterol is ergosterol.

In certain embodiments, the nutraceutical composition optionally comprises a probiotic wherein the probiotic is selected from a Bacillus sp., a Bifidobacterium sp., an Enterococcus sp., a Lactobacillus sp., a Lactococcus sp., a Pediococcus sp., a Saccharomyces sp., or a Streptococcus sp. In certain embodiments, the nutraceutical composition comprises a Bacillus sp. selected from Bacillus coagulans and Bacillus laterosporus. In certain embodiments, the nutraceutical composition comprises a Bifidobacterium sp. selected from Bifidobacterium breve, Bifidobacterium bifidum, Bifidobacterium infantis, Bifidobacterium lactis and Bifidobacterium longum. In certain embodiments, the nutraceutical composition comprises an Enterococcus sp. that is Enterococcus faecium. In certain embodiments, the nutraceutical composition comprises a Lactobacillus sp. selected from Lactobacillus acidophilus, Lactobacillus brevis, Lactobacillus bulgaricus, Lactobacillus casei, Lactobacillus fermentum, Lactobacillus gasseri, Lactobacillus helveticus, Lactobacillus infantis, Lactobacillus lactis, Lactobacillus plantarum, Lactobacillus paracasei, Lactobacillus rhamnosus, Lactobacillus reuteri and Lactobacillus salivarius. In certain embodiments, the nutraceutical composition comprises a Lactococcus sp. that is Lactococcus lactis. In certain embodiments, the nutraceutical composition comprises a Pediococcus sp. that is Pediococcus acidilactici. In certain embodiments, the nutraceutical composition comprises a Saccharomyces sp. that is Saccharomyces boulardii. In certain embodiments, the nutraceutical composition comprises a Streptococcus sp. that is and Streptococcus thermophilus. In particular embodiments, the nutraceutical composition comprises a Bacterioides sp. e.g. Bacteroides fragilis and Bacteroides acidifaciens; a Clostridium sp. e.g. Clostridium innocuum; a Peptoclostridium sp. e.g. Clostridioides difficile; or a Fusobacterium sp. e.g., Fusobacterium mortiferum.

In certain embodiments, an emulsion or microemulsion may be used for the encapsulation of spirulina. Encapsulation in the food industry is a process in which one or more ingredients or additives (core) are coated with an edible capsule. The use of liposomes and microemulsions are among the many forms of encapsulation of food ingredients. Encapsulation consists of a sort of microscopic lipid vesicles, where due to the lipophilic and hydrophilic portion of its constituents, substances of various natures can be encapsulated, and the hydrophilic substances stay in the aqueous compartment and the lipophilic components are inserted or adsorbed on the membrane or surface of the hydrophilic substance. Sonication and homogenization processes may be used in the encapsulation of a protein source, such as the cyanobacterium Spirulina platensis, formed out by the thin-film hydration method. Liposome or micelle emulsions and microemulsions may be prepared using an appropriate emulsifier such as phosphatidylcholine or microemulsifier such as Plural® Oleique and sonicated at, e.g. 60° C. for e.g. 30 min. In certain embodiments, a liposome emulsion may be prepared by homogenizing a phosphatidylcholine, or a microemulsion may be prepared by homogenizing a polyglycerol ester of oleic acid, at e.g. 10,000 rpm for e.g. 15 min. The average size, encapsulation efficiency, and particle morphology is then determined. Either sonication or homogenization may be used to obtain nanometric size particles. In certain embodiments, an emulsion or microemulsion is formed with homogenization. In certain embodiments, an emulsion or microemulsion is formed with sonication.

In certain embodiments, a component in the nutraceutical composition is encapsulated. In certain embodiments, the spirulina is encapsulated. In certain embodiments, the spirulina is encapsulated with an emulsion or with a microemulsion. In certain embodiments, the emulsion or microemulsion comprises a polyglycerol ester of oleic acid. In certain embodiments, the microemulsion comprises glyceryl dibehenate. In certain embodiments, the microemulsion comprises Compritol 888 ATO. In certain embodiments, the microemulsion comprises di- and triglyceride esters of fatty acids. In certain embodiments, the microemulsion comprises Gelucire 43/01. In certain embodiments, the emulsion or microemulsion comprises a phosphatidyl choline. In certain embodiments the emulsion or microemulsion comprises a beta-glucan.

In certain embodiments, the probiotic is encapsulated. In certain embodiments, the probiotic is encapsulated with a polysaccharide.

Compositions—Specific

TABLE 1

Four fecal samples from three members of a Severe Acute Malnutritian (SAM) trial

Age of

Time since
Anthropometry at time

Donor

discharge
of sample collection

Donor
(months)
Gender
from hospital
WHZ
WAZ
HAZ

1
19.2
M
5 months
−2.73
−2.71
−1.59

2
19.8
M
6 months
−1.78
−2.34
−2.23

3
18.1
M
5 months
−1.9
−2.18
−1.71

4
13.3
M
1 week prior
−3.27
−3.44
−2.35

TABLE 2A

(T2A) Mirpur-18 (M18) base diet

M18 base diet
Final weight (%)

Cooked rice
41.8

Whole milk powder
13.9

Cooked lentils
16.2

Cooked potato
6.9

Cooked spinach
6.3

Cooked Onion (yellow)
3.7

Soybean oil
3.7

Sweet pumpkin
6

Salt (iodized)
0.5

Turmeric
0.5

Garlic
0.5

Total
100

TABLE 2B

(T2B) Supplemented M18 Diet

Supplemented M18
Final weight (%)

Spirulina
5

Flaxseed
2.5

Amaranth
10

Micronutrients:
0.025

Folic acid-0.8 μg

Vitamin B3-30 μg

Vitamin B6-2.5 μg

Vitamin C-760 μg

Vitamin D-0.16 μg

Vitamin E-80 μg

Zinc-40 μg

Sub-total
17.525

(amaranth, flaxseed, spirulina,

rnicronutrients mix)

M18 Base
82.475

Total
100

In certain embodiments, a base diet comprises variations, by weight, of the any of the M18 ingredients described in Table 2A. In certain embodiments, for example, cooked rice amounts may range from about 30% to about 45%, milk powder may range from about 8% to about 20%, cooked lentils may range from about 10% to about 20%, cooked potato may range from may range from about 5% to about 10%, cooked spinach may range from about 5% to about 10%, cooked onion may range from about 2% to about 5%, soybean oil may range from about 2% to about 5%, sweet pumpkin may range from about 4% to about 10%, salt may range from about 0.25% to about 0.7%, turmeric may range from about 0.2% to about 1%, and garlic may range from about 0.2% to about 2%.

In certain embodiments, a base diet comprises variations by component, of any of the M18 ingredients described in Table 2A. In certain embodiments, for example, cooked rice may substituted with cooked barley, cooked quinoa, cooked wheat bulger, cooked oatmeal, cooked wheat bran, cooked tapioca and combinations thereof; milk powder may be substituted with buttermilk powder, coconut milk powder, soy milk powder, rice milk powder, potato milk powder, protein powder or combinations thereof; cooked lentils may be substituted with cooked chickpeas, cooked beans, cooked peas, almond paste, peanut butter or combinations thereof; cooked potato may be substituted with cooked turnip, cooked beets, cooked carrots or combinations thereof; cooked spinach may be substituted with cooked mustard greens, cooked kale, cooked turnip greens, cooked dandelion greens, cooked bok choy, cooked cabbage, cooked brussel sprouts, cooked broccoli or combinations thereof; cooked onion may be substituted with cooked leeks, cooked scallions, cooked cabbage or combinations thereof; soybean oil may be substituted with corn oil, coconut oil, peanut oil, sesame oil, rapeseed oil, sunflower oil, olive oil, canola oil, safflower oil, grapeseed oil, or combinations thereof; sweet pumpkin may be substituted with yams, various squash, carrots, beets or combinations thereof; salt may be iodized NaCl, rock salt such as Himalayan salt, sea salt or combinations thereof, turmeric may be substituted with ground ginger, curry powder, cumin, mustard powder, or combinations thereof; and garlic may be substituted with leeks, spring onions, scallions or combinations thereof.

In certain embodiments, a base diet comprises variations, by weight, and variations by component, of any of the M18 ingredients described in Table 2A. In certain embodiments, for example, Cooked rice amounts may range from about 30% to about 45%, milk powder may range from about 8% to about 20%, cooked lentils may range from about 10% to about 20%, cooked potato may range from may range from about 5% to about 10%, cooked spinach may range from about 5% to about 10%, cooked onion may range from about 2% to about 5%, soybean oil may range from about 2% to about 5%, sweet pumpkin may range from about 4% to about 10%, salt may range from about 0.25% to about 0.7%, turmeric may range from about 0.2% to about 1%, and garlic may range from about 0.2% to about 2%.