Immunomodulatory composition comprising bifidobacteria
10668117 ยท 2020-06-02
Assignee
Inventors
Cpc classification
International classification
A23L33/135
HUMAN NECESSITIES
Abstract
The present invention relates to composition derivable from a bifidobacteria, methods and uses thereof, for use in modulating the immune system in a subject by affecting viral action and/or viral effects in said subject. Furthermore, said composition may also be used in reducing the risk of development and exacerbation of chronic respiratory diseases such as asthma and COPD in a subject; preferably by modulating the immune system in the subject by affecting viral action and/or viral effects in said subject.
Claims
1. A method for reducing replication of a virus and/or inflammation caused by a virus infection in a subject in need thereof, wherein: the method comprises administering to the subject a composition comprising an effective amount of Bifidobacterium animalis subsp. Lactis BL-04 or a fermentation product or cell lysate thereof, and the virus comprises a rhinovirus or an influenza virus.
2. The method according to claim 1, wherein inflammation caused by the virus infection is reduced in the subject.
3. A method for reducing a level of one or more cytokines and/or chemokines caused by a virus infection in a subject in need thereof, wherein: the method comprises administering to the subject a composition comprising an effective amount of Bifidobacterium animalis subsp. Lactis BL-04 or a fermentation product or cell lysate thereof, and the virus comprises a rhinovirus or an influenza virus.
4. The method according to claim 3, wherein the method comprises modulation of the subject's innate immune system, adaptive immune system or both.
5. The method according to claim 3, wherein T-helper cells specific to the virus are increased in the subject.
6. The method according to claim 1, wherein the composition is formulated as a medicament, a food product or a dietary supplement.
7. The method according to claim 1, wherein said composition also comprises at least one further probiotic microorganism.
8. The method according to claim 1, wherein said composition also comprises one or more of an anti-microbial agent, a stabilising agent, a dye and a drying agent.
9. The method according to claim 1, wherein; the subject is at risk of developing a chronic respiratory disease, and the method reduces the risk.
10. The method according to claim 1, wherein the virus comprises a human rhinovirus.
11. The method according to claim 3, wherein IL-8, IL-1beta and/or MCP-1 is/are reduced in the subject.
12. The method according to claim 1, wherein the composition comprises an effective amount of Bifidobacterium animalis subsp. lactis BL-04.
13. The method according to claim 12, wherein the subject is a human.
14. The method according to claim 12, wherein IL-8 is reduced in the subject.
15. The method according to claim 12, wherein virus replication is reduced in the subject.
16. The method according to claim 12, wherein virus shedding is reduced in the subject.
17. The method according to claim 12, wherein the virus comprises an influenza virus.
18. The method according to claim 3, wherein the composition comprises an effective amount of Bifidobacterium animalis subsp. lactis BL-04.
19. The method according to claim 18, wherein a cytokine level is reduced in the subject.
20. The method according to claim 18, wherein IL-8 is reduced in the subject.
21. The method according to claim 18, wherein virus shedding is reduced in the subject.
22. A method for reducing virus shedding or an IL8 level in a virus-infected subject in need thereof, wherein: the method comprises administering to the subject a composition comprising an effective amount of Bifidobacterium animalis subsp. Lactis BL-04 or a fermentation product or cell lysate thereof, and the virus comprises a rhinovirus or an influenza virus.
23. The method according to claim 22, wherein the composition comprises an effective amount of Bifidobacterium animalis subsp. lactis BL-04.
24. The method according to claim 23, wherein IL-8 is reduced in the subject.
25. The method according to claim 23, wherein virus shedding is reduced in the subject.
26. The method according to claim 23, wherein; the subject is at risk of developing a chronic respiratory disease, and the method reduces the risk.
Description
FIGURES
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EXAMPLES
(11) The present invention will be further described with reference to the following Examples
Example 1Use of BL-04 in Healthy Adults
(12) Study Design and Flow
(13) Healthy adult volunteers were selected according to clinical study protocol to participate into the randomized, double-blind, placebo controlled study. Probiotic Bl-04 or placebo was administered for 28 days before inoculating the human rhinovirus (HRV) intranasally. The nasal lavages were taken daily up to 5 days post-infections to analyze the viral load and the inflammatory marker IL-8.
(14) This study was a randomized, double-blinded, placebo-controlled trial to assess the effects of an orally ingested probiotic on the host response to an experimental rhinovirus infection. The virus challenge day was Study Day 0. Healthy, young adult volunteers were started on Bl-04 or placebo on Study Day 28 after baseline blood and nasal lavage specimens had been collected. Subjects were instructed to consume one sachet of study product each day and were seen in the study center on study days 21, 14 and 7 to re-supply the study product and to assure compliance. The consumption of the study product was continued through Study Day 5.
(15) On Study Day 0, prior to virus inoculation, blood and nasal lavage was collected for study assays. The volunteers were then seen daily for the next 5 days to record symptoms and adverse events and to collect nasal lavage for viral culture.
(16) On study day 0, 5 and 21 blood was collected to analyse the adaptive immune cells by flow cytometry.
(17) Subjects:
(18) Seven hundred eighty-nine (789) subjects signed consent for participation in the trial. The study was conducted in three cohorts two in the spring of 2013 and one in the fall of 2013. The flow of subjects through the study is shown in
(19) One-hundred ninety (190) subjects were randomized to treatment and, after withdrawals and per protocol exclusions, 115, 58 in the active group and 57 in the placebo group, were available for analysis. The planned analysis sample size for the study was 120. The pivotal analysis for the study was done, as described in the statistical plan, on those subjects who were infected with rhinovirus, 51 subjects in the active group and 55 subjects in the placebo group. We note that there were a large number of exclusions form the study due to the consumption of prohibited foods (for example other probiotics) and voluntary withdrawal (See
(20) The study population was drawn primarily from the UVa student population and the demographics of the study reflect that population. The study arms were appropriately balanced for the demographics measured. Details of the demographics of a study subjects are given in table 1 below.
(21) TABLE-US-00001 TABLE 1 Study demographics. Active (N = 58) Placebo (N = 57) P value Gender Male 19 (33%) 24 (42%) 0.339 Female 39 (67%) 33 (57%) Race Asian 2 (3%) 8 (14%) 0.270 Black 3 (5%) 2 (4%) White 52 (90%) 46 (81%) Other 1 (2%) 1 (2%) Ethnicity Hispanic 3 (5%) 4 (7%) 0.857 Non-hispanic 54 (93%) 52 (91%) Unknown 1 (2%) 1 (2%) Age (yrs, mean (SD)) 22 (6) 23 (7) 0.975
(22) The effectiveness of the blinding of subjects to study treatment was assessed after the volunteers had consumed the study treatment for 28 days and before they were challenged with virus (see Table 2 below). The majority of volunteers in both treatment groups believed they were taking the placebo preparation but there was no evidence of unblinding (Likelihood Ratio Chi-square Test: P=0.56).
(23) TABLE-US-00002 TABLE 2 The assessment of blinding. Subject Guess Actual Group Active Placebo Don't know Active (N (%)) 11 (19.0) 31 (53.4) 16 (27.6) Placebo (N (%)) 7 (12.3) 31 (54.4) 19 (33.3)
(24) There were no serious adverse events during the study.
(25) Results
(26) The overall purpose of this study was to address the hypothesis that prophylaxis of human volunteers with probiotic bifidobacteria, preferably Bifidobacterium animalis, more preferably from Bifidobacterium animalis subsp. lactis, more preferably Bifidobacterium lactis BL-04 will inhibit virus-induced innate inflammatory responses characterized for example by IL-8, while enhancing the adaptive host response, for example antibody production. The study addressed the following specific outcome measures:
(27) Primary End-Point of the Clinical Study
(28) 1) Inflammatory Response in Nasal Mucosa as Characterized by IL-8 Levels in Nasal Lavages
(29) Nasal lavage IL-8 concentration was assessed by comparing the change from the baseline value in the concentration at 72 hours of observation (72 hr. measurements minus the 0 hr. measurement). The increase in IL-8 concentration between study day 0 and study day 3 was not significantly different between the probiotic and placebo groups (geometric mean ratio, active:placebo for change in concentration at day 3=0.69, p=0.14) (Table 3 line Day 3;
(30) TABLE-US-00003 TABLE 3 Baseline (day 28) adjusted between-group comparisons of the change in IL-8 concentration during infection from day 0, when expressed as a ratio of geometric means. Estimated Bonferroni Bonferroni Ratio of Lower Upper Un- Lower Upper Bonferroni Geometric 95% 95% adjusted 95% 95% Corrected Day Ratio Means CL CL Pvalue CL CL Pvalue 1 A:P 0.75 0.53 1.05 0.096 0.48 1.16 0.385 2 A:P 0.63 0.37 1.06 0.083 0.32 1.24 0.334 3 A:P 0.69 0.42 1.13 0.141 4 A:P 0.63 0.38 1.05 0.077 0.33 1.22 0.308 5 A:P 0.56 0.33 0.96 0.034 0.29 1.11 0.135 Overall A:P 0.65 0.44 0.95 0.026
(31) TABLE-US-00004 TABLE 4 Summary for the analysis of day 0 IL-8 concentration (loge[pg/mL]), after 28 days of probiotic supplementation. Estimate Lower Geometric 95% Upper 95% Study Mean IL-8 Confidence Confidence Arm Concentration Limit Limit Active 97.2 68.5 137.9 Placebo 58.0 43.4 77.5 Study Estimated Lower Pvalue Parameter Arm Geometric 95% Upper 95% Ho: Comparison Ratio Mean Ratio CL CL Ratio = 1 Geometric Active: 1.68 1.07 2.62 0.025 Mean Placebo
(32) TABLE-US-00005 TABLE 5 Unadjusted estimates for the change in IL-8 concentration during infection (days 1-5) from day 0 (i.e. baseline) when expressed as a ratio of geometric means. Estimated Lower Upper Pvalue Ho: Geometric 95% 95% Ratio = Study Group Ratio Mean Ratio CL CL 1 Active Day 1: 0.68 0.53 0.87 0.003 Day 0 Placebo 0.91 0.72 1.15 0.432 Active Day 2: 0.88 0.60 1.29 0.507 Day 0 Placebo 1.40 0.97 2.02 0.069 Active Day 3: 1.34 0.94 1.93 0.108 Day 0 Placebo 1.96 1.38 2.77 <0.001 Active Day 4: 1.16 0.80 1.68 0.417 Day 0 Placebo 1.85 1.30 2.63 0.001 Active Day 5: 0.91 0.62 1.33 0.622 Day 0 Placebo 1.62 1.12 2.33 0.011
Secondary End-Points
1, Biomarker Response of Nasal Mucosa
(33) Biomarker (cytokine and chemokine) profiles were measured from nasal lavages using multiplex ELISA assays. The following biomarkers were analyzed on days 28, 0, 1, 2, and 4 in relation to the virus challenge: TGF-beta1, G-CSF, GM-CSF, IFN-gamma, IL-1alpha, IL-12p70, IL-15, MIP-3alpha, IL-1beta, IL-6, IL-10, IP-10, MCP-1, MIP-1alpha, RANTES, and TNF-alpha (trimer). The concentrations of many of these analytes were too low to permit a valid analysis. The results for G-CSF, MIP-3alpha, IL-1beta, IL-6, IP-10, and MCP-1 were considered valid for analysis. Innate inflammatory markers were noted to increase after infection as expected. There were no significant differences between the treatment groups. However, there was a trend for a higher IL-1beta (p=0.071) and MCP-1 (p=0.075) response in the nasal lavages for a placebo group when a change in response from day 0 (infection) to day 4 was analyzed (
(34) TABLE-US-00006 TABLE 6 Active Placebo Active Active GM GM vs. Active GM Ratio Placebo Ratio Placebo Day 0 Placebo Pvalue Ratio (Day GM (Day 4: (Day 4: GM Day 0 Active (Day 4: 4:Day Ratio Day Day [95% GM vs. Day 0) (Day 4: 0) 0) Cytokine CI] [95% CI] Placebo 0) Pvalue Day 0) Pvalue Pvalue G-CSF 33.4 20.4 0.155 3.35 <0.001 4.87 <0.001 0.336 [20.1, [12.7, [1.95, [2.78, 55.4] 32.6] 5.73] 8.54] IL-1beta 0.9 0.6 [0.4, 0.250 1.04 0.889 2.13 0.007 0.071 [0.6, 1.5] 1.0] [0.54, [1.24, 1.85] 3.69] IL-6 5.5 3.9 [2.6, 0.283 3.73 <0.001 5.56 <0.001 0.331 [3.5, 8.6] 5.9] [2.12, [3.08, 6.57] 10.05] IP-10 439.1 298.8 0.144 4.10 <0.001 6.24 <0.001 0.236 [317.0, [198.2, [2.57, [3.66, 608.3] 450.6] 6.53] 10.62] MCP-1 12.9 12.2 [9.5, 0.757 1.02 0.878 1.55 0.002 0.075 [9.8, 17.0] 15.6] [0.71, [1.18, 1.48] 2.02]
2. Rhinovirus Replication
(35) Rhinovirus quantity was measured in nasal lavage by standard culturing methods. Nasal lavage viral titers were generally greater in the placebo-treated than in the probiotic-treated subjects, however, this difference was not statistically significant (p=0.1) (Table 7,
(36) TABLE-US-00007 TABLE 7 Degrees Wald Chi-square Source of Variation of Freedom Statistics Pvalue Study-Arm 1 2.68 0.101 Study-Day 4 97.37 <0.001 Study-Arm Study 4 7.44 0.114 Day
(37) TABLE-US-00008 TABLE 8 Degrees Wald Chi-square Source of Variation of Freedom Statistics Pvalue Study-Arm 1 4.92 0.026 Study-Day 4 93.01 0.000 Study-Arm Study 4 8.95 0.062 Day
(38) The number of circulating RV-specific T-cells in the subset of DR4+ subjects: Two HRV39 peptide/MHC II tetramers will be used for direct ex vivo quantitative analysis of circulating HRV39-specific CD4+ T cell frequencies using PBMC specimens. It was found that fold change in HRV39-specific CCR5+T-helper cells was greater in probiotic group at day 21 (p=0.042) (
(39) The results for
(40) TABLE-US-00009 Lower Upper Bonferroni Bonferroni Bonferroni Fold 95% 95% Unadjusted Lower Upper Corrected Day Ratio Change CL CL P Value 95% CL 95% CL P Value 5 Probiotic: 1.24 0.75 2.06 0.390 0.69 2.23 0.780 Placebo 21 1.84 1.11 3.05 0.021 1.02 3.31 0.042
(41) The results indicate that adaptive immune response against HRV39 was improved after the infection and Bl-04 supplementation could thus provide benefit against reinfections of HRVs.
(42) The results of Example 1 show that the prophylactic use of Bl-04 in an experimental rhinovirus infection model decreased the inflammatory IL-8 cytokine levels in nasal lavage. The reduced inflammatory response in HRV infection by Bl-04 was supported by the trend for higher induction of inflammatory cytokines IL-1beta and MCP-1 in placebo group, but not in the probiotic group. These results indicate that consumption of Bl-04 may alleviate the inflammatory response in the airways. In addition, the results above show that Bl-04 consumption is effective against HRV replication in healthy subjects. This also indicates that Bl-04 could prevent asthma development in infants and/or children and be beneficial in preventing asthma and COPD exacerbations that are associated with rhinovirus infections.
Example 2Chronic Respiratory Diseases
(43) Introduction
(44) The above study demonstrates that probiotic B. lactis Bl-04 decreases rhinovirus shedding and IL-8 levels during HRV infection in humans indicating an anti-viral and anti-inflammatory effect, respectively, by the probiotic.
(45) Without wishing to be bound by theory, we believe that the administration of Bl-04 could decrease risk (anti-viral effect) or severity (anti-inflammatory effect) of asthma or COPD exacerbations. To test this, mouse model that has been made susceptible for human rhinoviruses (Bartlett et al. 2008) is used to test the efficacy of Bl-04 supplementation against asthma or COPD exacerbations.
(46) Asthma is studied in mice that are sensitized to ovalbumin, a model allergen, and COPD is studied in mice that are exposed chronically to cigarette smoke (Stevenson and Birrell 2011). Optionally probiotic Bl-04 is administered prophylactically in experimental rhinovirus challenge model using asthmatic or COPD human subjects that have mild and stable disease (Del Vecchio et al. 2015; Contoli et al. 2007; Cheung et al. 1995).
(47) Animal Studies
(48) Animal Model for HRV Infection
(49) The effect of the Bl-04 supplementation against rhinovirus is studied in mice by using minor group rhinovirus infected mice or mice transgenic for human ICAM-1 receptor that acts as a natural receptor for human major group rhinoviruses in humans (e.g. Bartlett 2008 Nat Med). This model is used to study the effect Bl-04 against HRV infection. When combined with other specific models, explained below, (i) reduction of asthma development risk, (ii) reduction of asthma exacerbation risk, and for (iii) reduction of COPD exacerbation risk may be studied.
(50) Dosing of Probiotic in Animal Experiments
(51) Bl-04 is preferably fed at dose of 2*10.sup.9 CFU/day for 2 weeks, before HRV infection. The probiotic is administered either by gavage or mixed into small amount of feed or liquid to assure that the dose is ingested.
(52) The Effect of the Probiotic Bl-04 on Asthma Development
(53) The HRV susceptible mice are administered Bl-04 or placebo for 1 week before exposing them to HRV, RSV, or placebo infection at day 7 post-natal (Schneider 2012). The challenge is done at 50% infective dose. Rationale for RSV inclusion is that Bl-04 could be potentially effective against other asthma predisposing viruses than HRV as well (Schwarze et al. 1997). RSV causes wheezing more commonly then HRV. We monitor the inflammation of the airways and virus titers in mice during the infection. Bl-04 has antagonistic effects on HRV and IL-8 levels.
(54) After the HRV infection, the mice are further sensitized to model antigen ovalbumin (OVA) that can be used to induce asthma like symptoms in airways by three consecutive doses using alum as an adjuvant. The inflammation of the airways and development of airway hyper-responsiveness are monitored by standard histological and molecular methods. The early life or post-natal, Bl-04 supplementation and decrease in HRV pathology and inflammation during infection decrease inflammation and asthma pathology in adult mice later in their life.
(55) The Effect of the Probiotic Bl-04 on Asthma Exacerbations
(56) The HRV susceptible mice are sensitized to ovalbumin with alum adjuvant or PBS as a control. OVA is administered on three consecutive days to induce airway inflammation. The supplementation of the HRV susceptible mice is started with Bl-04 or placebo as described above. After 2 weeks of Bl-04 supplementation, a HRV challenge at 50% infective dose is given to mice and inflammation (e.g. IL-8) of the airways and rhinovirus titers in the airway lavages are analyzed from mice. Furthermore, airway pathology is monitored histologically. In the Bl-04 group the inflammation, HRV titer, and pathology are alleviated and potentially the infectivity of HRV is decreased.
(57) The Effect of the Probiotic Bl-04 on COPD Exacerbations
(58) The HRV susceptible mice are exposed to cigarette smoke for at least 1 month (or not in the case of the placebo group), to induce immunological and pathological changes observed in COPD (Stevenson 2007). The supplementation of the mice with Bl-04 or placebo is initiated for 2 weeks after chronic COPD like changes have occurred. Then the mice are inoculated with 50% infective dose of HRV to induce acute virus induced exacerbation. Inflammation (incl. IL-8), HRV titers and lung pathology are analyzed. In the Bl-04 group, the inflammation, HRV titer, and pathology are alleviated and potentially the infectivity of HRV is decreased.
Example 3Effect of Bl-04 on Asthma Exacerbation
(59) Study Design
(60) Treatment Groups:
(61) 1. Negative control group. Treatment with vehicle (H.sub.2O).
(62) 2. Treatment with Bl-04.
(63) Number of mice per group=15.
(64) House-Dust-Mite (HDM) Induced Asthma Exacerbation:
(65) Day 0-28: Three times per week administration of HDM in 30 ul PBS per nasal.
(66) Day 28-49: Daily administration of Bl-04 or vehicle (H.sub.2O) by cereal treat.
(67) Day 49: Administration of sub-lethal dose of influenza virus (PR8) per nasal.
(68) Day 52: 5 animals per group are sacrificed for analysis.
(69) Day 54: 5 animals per group are sacrificed for analysis.
(70) Day 56: 5 animals per group are sacrificed for analysis.
(71) 8 week old BALB/c mice were used for the study. Potable water and food were available ad libitum.
(72) At days 0 to 28, animals were administered three times per week with 15 g of HDM in a volume of 30 l PBS per nasal. Mice were anesthetized by intraperitoneal injection with 9.75 mg Xylasol and 48.75 mg Ketasol per kg and administered with 15 g of HDM in a volume of 30 l PBS per nasal.
(73) At days 28 to 49, animals were administered with vehicle (water, Group 1) or Bl-04 (Group 2) by oral gavage (110.sup.9 CFU/day) and by cereal treat (110.sup.9 CFU/day/cereal.
(74) 200 l of water or Bl-04, reconstituted in water at a concentration of 510.sup.9 CFU/ml, was placed on a single flake of cereal (Special K manufactured by Kellogg's). The probiotic treatments were prepared fresh each day of administration. One flake of cereal per mouse in each cage was placed on a feeding tray inside the cage and the mice were able to eat during the day.
(75) On the same day, 200 l of water or the reconstituted probiotic was administered to mice by oral gavage.
(76) On day 49, mice were inoculated with a sub-lethal dose of influenza A virus (250 PFU per nasal; influenza strain H1N1 PR/8/34)). The virus material was stored at 75 C.10 C. and was defrosted prior to administration. Once defrosted, the material was diluted in cold PBS (4 C.) corresponding to 250 PFU/50 l for A/PR/8/34. The diluted virus was kept on ice until administration to the mice.
(77) The animals were anaesthetized by intraperitoneal injection with 9.75 mg Xylasol and 48.75 mg Ketasol per kg body weight and each animal received 50 l virus solution by intranasal inoculation.
(78) On days 52, 54 and 56 five animals per group were sacrificed by lethal intraperitoneal injection with pentabarbitol immediately followed by a bronchoalveolar lavage (BAL) in 500 ul of saline and tissue isolation (lung).
(79) Cells were isolated from the BAL fluid and differential cell counts performed (200 cell counts/samples). The total cell numbers in the bronchoalveolar lavage (BAL) fluid was determined using a Coulter Counter (IG Instrumenten-Gesellschaft AG, Basel, Switzerland). Differential cell counts were performed (200 cell counts/samples) based upon standard morphological and cytochemical criteria on cytospins stained with Diff-Quik solution (Dade Behring, Siemens Healthcare Diagnostics, Deerfield, Ill.).
(80) Lung lobes were isolated for the quantification of viral load in lung tissue by quantitative PCR. Lung lobes were carefully isolated and placed in 2 ml Eppendorf tubes containing 1 ml of TRI-Reagent (Molecular Research Centre Inc. Catalogue number: TR 118) and snap frozen in dry ice. Samples were then stored at 80 C. until processing.
(81) Lung lobes preserved in TRI-Reagent, were slowly thawed at room temperature. Sterile, stainless steel beads (Qiagen. Catalogue Number: 69989) were placed in each sample tube and lungs were homogenised using a TissueLyser (Qiagen. Catalogue Number: 85220), at a frequency of 25 Hz for 3 minutes.
(82) RNA extraction using TRI-Reagent (Molecular Research Centre Inc. Catalogue number: TR 118) was completed following manufacturer's instructions (http://mrcgene.com/wp-content/uploads/2014/06/TRIMay2014.pdf).
(83) RNA concentration in each sample was measured using a Thermo Scientific NanoDrop 1000 Spectrophotometer (Thermo Fisher Scientific). RNA was treated with DNase (Invitrogen) to avoid genomic DNA contamination before RNA was converted to cDNA by reverse transcription and cDNA was quantified by real-time PCR. Samples were normalized with reference to -actin expression levels.
(84) Samples were amplified in a one-step RT-PCR at a final reaction volume of 10 l, containing 5 l of iTaq universal SYBR Green reaction mix (BIO-RAD Laboratories, Catalogue number 172-5150), 0.125 l of iScript reverse transcriptase (BIO-RAD. Catalogue number: 172-5150), 0.5 l of Influenza PR8 Matrix protein forward and reverse primers (5_-GGACTGCAGCGTAGACGCTT-3_ and 5_-CATCCTGTATATGAGGCCCAT-3) or Beta actin (5_-GATCAAGATCATTGCTCCTCCTGA-3_ and 5_-CAGCTCAGTAACAGTCCGCC-3_), 0.375 l of RNAse free water (BIO-RAD. Catalogue number: 172-5150), and 4 l of RNA at 125 ng/l.
(85) Thermo-cycling was performed in a CFX-96 real-time PCR system (BIO-RAD) using the following protocol: Reverse transcription reaction: 10 min at 50 C., then polymerase activation and DNA denaturation: 1 min at 95 C., then denaturation: 95 C. for 10 sec, then annealing/extension+ plate read: 30 sec at 60 C., for total of 40 cycles.
(86) Graphs and Statistical Analysis.
(87) Statistics were performed as either an unpaired t-test (quantification of viral titer in lung tissue) or as one-way ANOVA. Error bars in the figures represent Standard Error of the Mean (SEM).
(88) Results
(89) The administration of the probiotic was well tolerated by the mice. The mice received the probiotic by gavage, which ensured all mice were exposed to the bacteria, and additionally cereal in the cage was soaked with the probiotic and rapidly eaten by the mice each day. No substantial variation was noted within groups that would indicate that some mice consumed more of the bacteria-soaked cereal than other mice.
(90) Treating the mice with HDM introduced asthma-like conditions in the mice. After treatment with probiotics or vehicle the mice were infected with influence virus. The appearance of the probiotic treated mice was strikingly different, with the probiotic mice appearing to have less morbidity. This observation suggests that the probiotic treated mice might have been protected against the initial allergic asthma response, and/or the probiotic bacteria protect against morbidity (and potentially mortality) during viral infection. Thus, the overall well-being of the mice seems to be increased (
(91) The primary measurements of the immune response against influenza infection in mice that had asthma showed a tendency that Bl-04 could modulate immune response in the lungs during the influenza infection. The main immune cell constituents of the airways were quantified by microscopy, i.e. lymphocytes, macrophages and neutrophils. The number of macrophages on day 56 and the number of lymphocytes in the airways on day 54 were significantly higher for mice treated with probiotics than for mice receiving vehicle (
(92) Results from the quantitative measurements of viral load in the lung tissue can be seen in Table 9 below and in
(93) TABLE-US-00010 TABLE 9 Mouse beta actin beta actin beta actin Flu Flu Flu Flu/beta actin Flu/beta actin # Treatment Cq 1 Cq 2 Cq average Cq 1 Cq 2 Cq average Delta Cq ratio (Cq) 1.1 H2O, day 52 19.83 19.38 19.60 24.33 24.17 24.25 4.65 6.51E+03 1.2 H2O, day 52 20.30 20.15 20.23 26.33 26.19 26.26 6.04 2.88E+03 1.3 H2O, day 52 20.58 20.20 20.39 25.74 25.42 25.58 5.19 4.73E+03 1.4 H2O, day 52 19.09 18.76 18.93 23.48 23.31 23.40 4.47 7.24E+03 1.5 H2O, day 52 20.57 20.01 20.29 27.52 26.95 27.23 6.94 1.69E+03 2.1 Probiotics, day 52 20.02 19.85 19.94 25.07 24.82 24.94 5.01 5.27E+03 2.2 Probiotics, day 52 19.98 19.39 19.68 27.02 26.50 26.76 7.07 1.56E+03 2.3 Probiotics, day 52 18.84 18.43 18.64 23.30 22.96 23.13 4.49 7.14E+03 2.4 Probiotics, day 52 20.25 19.85 20.05 28.76 27.98 28.37 8.32 7.51E+02 2.5 Probiotics, day 52 20.53 20.06 20.30 25.40 25.24 25.32 5.02 5.23E+03 1.6 H2O, day 54 20.20 19.65 19.93 23.68 23.19 23.44 3.51 1.27E+04 1.7 H2O, day 54 20.09 19.96 20.03 24.62 24.34 24.48 4.46 7.29E+03 1.8 H2O, day 54 20.03 19.55 19.79 23.23 22.85 23.04 3.24 1.48E+04 1.9 H2O, day 54 19.15 18.90 19.02 22.42 22.47 22.45 3.42 1.34E+04 1.10 H2O, day 54 18.93 18.75 18.84 21.39 21.18 21.28 2.44 2.38E+04 2.6 Probiotics, day 54 20.49 20.25 20.37 24.40 24.26 24.33 3.96 9.77E+03 2.7 Probiotics, day 54 19.51 19.04 19.27 22.74 22.22 22.48 3.21 1.52E+04 2.8 Probiotics, day 54 19.84 19.47 19.66 23.38 23.09 23.23 3.57 1.22E+04 2.9 Probiotics, day 54 19.84 19.60 19.72 23.69 23.49 23.59 3.87 1.03E+04 2.10 Probiotics, day 54 19.84 19.30 19.47 23.32 23.14 23.23 3.76 1.10E+04 1.11 H2O, day 56 20.33 20.05 20.19 24.30 23.99 24.15 3.95 9.80E+03 1.12 H2O, day 56 21.26 21.01 21.13 26.49 26.21 26.35 5.22 4.65E+03 1.13 H2O, day 56 19.80 19.54 19.67 24.28 24.15 24.22 4.54 6.92E+03 1.14 H2O, day 56 19.34 19.11 19.22 26.58 26.51 26.54 7.32 1.35E+03 1.15 H2O, day 56 19.94 19.49 19.71 24.45 24.13 24.29 4.57 6.81E+03 2.11 Probiotics, day 56 18.80 18.37 18.58 24.23 23.73 23.98 5.40 4.19E+03 2.12 Probiotics, day 56 19.72 19.32 19.52 24.10 23.71 23.90 4.38 7.60E+03 2.13 Probiotics, day 56 19.25 19.04 19.15 24.53 24.20 24.37 5.22 4.66E+03 2.14 Probiotics, day 56 19.08 18.72 18.90 24.49 24.11 24.30 5.40 4.19E+03 2.15 Probiotics, day 56 18.05 17.67 17.86 23.68 23.43 23.55 5.69 3.53E+03
(94) The human clinical study in healthy adults suggested that Bl-04 could modulate innate immune response (measured as IL-8) that may have suppressed the rhinovirus shedding in the nasal mucosa. In the HDM mouse model supportive evidence on the effect of Bl-04 on immune modulation was observed as increased numbers of macrophages and lymphocytes were detected in the BALF. These immunomodulatory changes could explain the improved well-being of mice and potentially lower viral titer in the lungs of the mice that are in line with the decreased rhinovirus titer in the humans. Thus Bl-04 seems to improve anti-viral immunity against rhinovirus in healthy adults and influenza in asthmatic mice. Overall, the results suggest that Bl-04 could be applied to asthmatic humans for preventing or alleviating virus induced asthma exacerbations.
Example 4Human Clinical Studies
(95) Exacerbations of Asthma and COPD in Adults
(96) A protocol similar to described in this invention on experimental rhinovirus infection model in Example 1 is used to study the effect of Bl-04 supplementation on exacerbations of asthma and COPD (Del Vecchio 2015). Instead of healthy subjects asthmatics and COPD patients with mild to moderate disease are used by using standard clinical criteria.
(97) The Effect of Bl-04 on Preventing and Alleviating Asthma Exacerbations Induced by HRV
(98) Study Product
(99) Probiotic Bl-04 or placebo is administered at dose of 210.sup.9 CFU/day mixed into a drink for 28 days prior to HRV challenge and 4 days post infection.
(100) Study Design
(101) Randomized, double-blind, placebo controlled
(102) Study Endpoints
(103) Airflow obstruction of the subjects is measured using peak expiratory flow rate (PEF).
(104) This is a person's maximum speed of expiration, as measured with a peak flow meter. Also Forced Expiratory Volume in one second of the predicted (FEV1% predicted) is measured or any other airflow measures needed. PEF and FEV1% predicted are measured at days 28, 0, 1, 2, and at the end of the study on day 4.
(105) Airway inflammation is measured using the eNO measurement as per Example 1, and also by measuring IL-8 in bronchoalveolar lavage. These measurements are taken at days 28, 0, 1, 2, and 4.
(106) Respiratory symptoms are measured using a survey at days 28, 0, 1, 2, 3 and 4 (preferably using a questionnaire and FEV).
(107) Viral titer in bronchoalveolar and nasal lavage is measured as per Example 1.
(108) Inclusion Criteria
(109) Mild or moderate asthma based on PEF, FEV1% predicted and frequency of symptoms (Yawn. 2008)
(110) 18-65 years of age
(111) Not pregnant
(112) Exclusion Criteria
(113) Use of anti-inflammatory drugs
(114) Other chronic disease
(115) Regular probiotic consumption
(116) Antibiotic treatment
(117) The Effect of Bl-04 on Preventing and Alleviating COPD Exacerbations Induced by HRV
(118) An identical study as per the asthma study described above is carried out, but in this case, rather than asthma the subjects have mild or moderate COPD Vestbo. 2013)
(119) Development of Childhood Asthma
(120) The Effect of Bl-04 on Preventing Childhood Asthma
(121) Pregnant mothers who have a tendency for atopy (that is, allergic hypersensitivity which is known to be associated with an increase in the likelihood of developing asthma (Elward et al. 2010) and their babes are recruited into study. The probiotic Bl-04 is administered to babies from birth up to 12 years of age. The incidence of viral infections during the first year of life is monitored by visits to pediatricians and the etiology is discovered by standard laboratory techniques. In addition the development of the immune system and incidence atopy, wheezing and asthma is monitored up to 3 yrs of age by analyzing the Th2 cytokines, IgE, and using PEF, FEV1%, skin prick tests, and potentially other diagnostic measures for atopy and asthma.
(122) Study Product
(123) Probiotic Bl-04 or placebo is administered at dose of 210.sup.9 CFU/day mixed into a milk starting from birth and up to 12 months of age.
(124) Study Design
(125) Randomized, double-blind, placebo controlled
(126) Study Endpoints
(127) In addition to Airflow obstruction measured as described above, the following measurements are made:
(128) Skin prick tests (measurement of skin reaction to allergens)
(129) Respiratory symptoms are recorded by the parents using a questionnaire
(130) The incidence of HRV and RSV infections are measured by parental and physician survey.
(131) Inclusion Criteria
(132) Atopic tendency of parents
(133) Healthy newborns
(134) Exclusion Criteria
(135) Antibiotic treatment
(136) Other congenital anomalies at birth
(137) All publications mentioned in the above specification are herein incorporated by reference. Various modifications and variations of the described methods and system of the invention will be apparent to those skilled in the art without departing from the scope and spirit of the invention. Although the invention has been described in connection with specific preferred embodiments, it should be understood that the invention as claimed should not be unduly limited to such specific embodiments. Indeed, various modifications of the described modes for carrying out the invention which are obvious to those skilled in microbiology, biochemistry and molecular biology or related fields are intended to be within the scope of the following claims.
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