ALLERGY TREATMENT

Abstract

The present invention concerns a method of treating peanut allergy in a subject, or a population of subjects, the method comprising administering to said subject or population of subjects a peanut allergen by an oral immunotherapy (OIT) regimen comprising a dose escalation phase in which the peanut allergen is administered in a dose which is increased in increments from an initial dose of the allergen equivalent of 5 mg peanut protein or less to a dose of the allergen equivalent of 200 mg peanut protein or more within 4 to 9 weeks from the administration of the initial dose. The treatment reduces the length of the build-up phase in peanut OIT methods and improves the likelihood or odds of the subject achieving sustained unresponsiveness.

Claims

1. A method of treating peanut allergy in a subject, the method comprising administering to said subject a peanut allergen by an oral immunotherapy regimen comprising a dose escalation phase, wherein the peanut allergen is administered in a dose which is increased in increments from an initial dose of the allergen equivalent of 5 mg peanut protein or less, preferably an initial dose of the allergen equivalent of 2 mg peanut protein or less, to a dose of the allergen equivalent of 200 mg peanut protein or more within 4 to 9 weeks from administration of the initial dose.

2. The method of claim 1, wherein during said dose escalation phase the peanut allergen dose is increased in increments to (i) a dose of the allergen equivalent of 400 mg peanut protein or more within 6 to 11 weeks from administration of the initial dose, and/or (ii) a dose of the allergen equivalent of 800 mg peanut protein or more within 8 to 13 weeks from administration of the initial dose, and/or (iii) a dose of the allergen equivalent of 2 g peanut protein or more within 16 to 24 weeks from administration of the initial dose.

3. The method of claim 1, wherein the dose escalation phase comprises a series of roughly doubling dose increments from the initial dose to the dose of 200 mg, 400 mg, 800 mg, 2 g or more.

4. The method of claim 1, wherein the dose escalation phase is 8 to 24 weeks and a final dose is reached at the end of the dose escalation phase.

5. The method of claim 1, to wherein a final dose is reached at the end of the dose escalation phase and said treatment further comprises a maintenance phase following completion of the dose escalation phase, in which the peanut allergen is administered at a set maintenance dose which is ±10% of the final dose reached at the end of the dose escalation phase, preferably wherein (i) the maintenance dose is the allergen equivalent of at least 200 mg, preferably at least 400 mg, preferably at least 800 mg, particularly preferably at least 2 g, especially preferably about 200 mg, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, 2000, 2100, 2200, 2300, 2400, 2500, 2600, 2700, 2800, 2900, 3000, 3100, 3200, 3300, 3400, 3500, 3600, 3700, 3800, 3900, 4000, 4100 or 4200 mg peanut protein, and/or (ii) the duration of the maintenance phase is 2 weeks or more following the completion of the dose escalation phase, preferably wherein the duration of the maintenance phase is 20-104 weeks.

6. The method of claim 1, wherein: the peanut allergen dose is administered at least every 3 days, preferably every 2 days during the dose escalation and/or maintenance phase, preferably wherein the peanut allergen dose is administered every day.

7. The method of claim 1, wherein the dose escalation phase comprises a rush phase in which the peanut allergen is administered in a dose which is increased in increments from the initial dose to a dose of the allergen equivalent of 12 mg peanut protein or more within 1 hour to 1 week from administration of the initial dose, preferably wherein the rush phase is completed in 1 to 12 hours from administration of the initial dose.

8. The method of claim 1, wherein (i) the peanut allergen is, or is present in, or derived from, a peanut protein, and/or (ii) the peanut allergen is provided as peanut protein, preferably as peanut flour or a defatted form thereof, and/or (iii) the peanut allergen is one or more of Ara h1 to Ara h9 or a modified form thereof, preferably one or more of Ara h1, h2, h3, h6, h8 or h9, preferably Ara h2 or is a modified form thereof.

9. The method of claim 1, wherein the peanut allergen is administered in or on apple sauce, pudding, yoghurt or chocolate.

10. The method of claim 1, wherein the subject is less than 18 years old, preferably less than 10 years old, preferably less than 5 years old, wherein the subject is from 1 month to 12 years old, preferably wherein the subject is less than 6 years old or from 6 to 12 years old or 1 to 5 years old.

11. The method of claim 1, wherein reducing time to reach maintenance dose compared to time taken to reach a maintenance dose in an oral immunotherapy regimen using a longer dose escalation phase to reach a same maintenance dose improves the likelihood or the odds of the subject achieving sustained unresponsiveness, wherein preferably reaching the maintenance dose in 8 to 24 weeks improves the likelihood or the odds of the subject achieving sustained unresponsiveness compared to oral immunotherapy regimens using a longer dose escalation phase to reach the same maintenance dose.

12. A method of treating peanut allergy in a population of subjects, the method comprising administering to a populations of subjects a peanut allergen by an oral immunotherapy regimen comprising a dose escalation phase in which the peanut allergen is administered in a dose which is increased in increments from an initial dose of the allergen equivalent of 5 mg peanut protein or less to a dose of the allergen equivalent of 200 mg peanut protein or more within 4 to 9 weeks from administration of the initial dose and wherein preferably said peanut allergen, subject and/or oral immunotherapy regimen is as defined in claim 1, preferably wherein: a) said dose escalation from the allergen equivalent of 5 mg peanut protein or less to the allergen equivalent of 200 mg peanut protein or more is achieved within 4 to 9 weeks from administration of the initial dose in more than 75% of the population; or b) the median time to achieve said dose escalation from the allergen equivalent of 5 mg peanut protein or less to the allergen equivalent of 200 mg peanut protein or more is 4 to 9 weeks from administration of the initial dose.

13. The method of claim 1 or claim 12 further comprising administration of a probiotic orally at least once every week during said peanut dose escalation phase, preferably at a dose of 1×10.sup.8 cfu to 2×10.sup.10 cfu.

14. The method of claim 13 wherein the probiotic is administered at least every three days, preferably wherein the probiotic is to be administered daily.

15. The method of claim 14, wherein the dose escalation phase comprises a rush phase in which the peanut allergen is administered in a dose which is increased in increments from the initial dose to a dose of the allergen equivalent of 12 mg peanut protein or more within 1 hour to 1 week from administration of the initial dose, preferably wherein the rush phase is completed in 1 to 12 hours from administration of the initial dose, wherein the probiotic is administered at least once during the rush phase and at least once every week thereafter, preferably at a dose of 1×10.sup.8 cfu to 2×10.sup.10 cfu.

16. The method of claim 13, wherein the probiotic is capable of inducing an increased number of tolerogenic dendritic cells after in vivo administration, preferably wherein the tolerogenic dendritic cells are plasmacytoid dendritic cells (pDCs).

17. The method of claim 13, wherein the probiotic is a species of Lactobacillus, Bifidobacterium, Saccharomyces, Streptococcus, Escherichia or Bacillus, preferably wherein i) the probiotic is a species of Lactobacillus, selected from the list of Lactobacillus acidophilus, Lactobacillus casei, Lactobacillus johnsonii, Lactobacillus lactis, Lactobacillus plantarum, Lactobacillus reuteri, Lactobacillus rhamnosus, Lactobacillus salivarius and Lactobacillus helveticus, preferably wherein the probiotic is Lactobacillus rhamnosus, or ii) the probiotic is Lactobacillus rhamnosus GG, Bifidobacterium lactis, Saccharomyces boulardii, Escherichia coli Nissle 1917, Streptococcus thermophilus or Bifidobacterium breve.

18. The method of claim 13, wherein for each administration of peanut allergen, the probiotic and peanut allergen are administered simultaneously, separately or sequentially, preferably simultaneously in a single or separate compositions, preferably wherein for each administration of peanut allergen, the peanut allergen is administered one minute, two minutes, three minutes, four minutes, five minutes, ten minutes, 30 minutes, 1 hour, or 2 hours or more up to 12 hours before the probiotic, or, the probiotic is administered one minute, two minutes, three minutes, four minutes, five minutes or ten minutes, 30 minutes, 1 hour, or 2 hours or more up to 12 hours before the peanut allergen.

19. The method of claim 13, wherein for each administration of peanut allergen, the peanut allergen and probiotic are administered separately up to 7 days apart, preferably up to 1, 2, 3, 4, 5 or 6 days apart.

20. The method of claim 13, wherein the probiotic is administered in water, cow's milk, soy milk, or other milk or drink.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0193] The invention will now be described by way of non-limiting Examples with reference to the drawings in which:

[0194] FIG. 1 shows the time in weeks to reach the 2000 mg peanut protein maintenance dose (FIG. 1A) and the percentage of time the build-up phase extended longer than the expected 32 weeks for the standard build-up schedule and the expected 16 weeks for the rapid build-up schedule (FIG. 1B) with medians and interquartile ranges for the standard build-up schedule compared with the rapid build-up schedule. * P<0.05, **** P<0.0001. The Mann-Whitney test was used to determine statistical differences.

[0195] FIG. 2 shows the number of delayed build-up visits (FIG. 2A), percentage of delayed build-up visits (FIG. 2B), and delayed days on dose (FIG. 2C) with medians and interquartile range for the standard build-up schedule and the rapid build-up schedule. * P<0.05, ** P<0.01, P<0.0001. The Mann-Whitney test was used to determine statistical differences.

[0196] FIG. 3 plots cumulative dose taken during build-up (FIG. 3A) and average dose taken each week (FIG. 3B) with medians and interquartile ranges. * P<0.05, **** P<0.0001. The Mann-Whitney test was used to determine statistical differences.

[0197] FIG. 4 shows the number of participants with moderate and severe adverse events (AEs) during rush day and build-up phase in the standard build-up schedule and the rapid build-up schedule. The different shadings show how many participants experienced a certain number of moderate to severe AEs.

[0198] FIG. 5 shows the proportions of pDCs in adults with (n=58) and without (n=62) allergic disease before and after 14 days of treatment with probiotics. The Mann-Whitney test was used to determine statistical differences between subjects with and without allergic disease and the Wilcoxon matched-pairs signed rank test was used to determine the statistical differences between subjects assessed on day 0 or day 14.

EXAMPLES

Example 1—Comparison of Outcomes for a Protocol with Standard Time to Maintenance Dose (PPOIT-001) and a Protocol with Reduced Time to Maintenance (PPOIT-002)

[0199] In this Example peanut OIT protocols were compared in which a standard administration protocol (PPOIT1) and a protocol in which the time to maintenance dose of OIT was reduced (PPOIT2) were used.

Materials and Methods

Study Population

[0200] In the PPOIT-001 trial, 62 peanut allergic children, aged 1 to 10 years, were enrolled to the randomized, double-blind placebo controlled trial which combined the probiotic Lactobacillus rhamnosus GG (single dose of 2×10.sup.10 cfu taken daily just before ingestion of peanut OIT) and peanut OIT for a total of 18 months, the first 8 months consisting of the dose-escalation phase comprising a rush day followed by a build-up phase (time to maintenance 32 weeks). In PPOIT-002, 20 peanut allergic children, were enrolled in an open pilot study of the probiotic Lactobacillus rhamnosus GG (single dose of 2×10.sup.10 cfu taken daily before ingestion of peanut OIT) and peanut OIT where the dose escalation phase was shortened to 4 months (time to maintenance 16 weeks).

[0201] Children enrolled in PPOIT-001 study were 1 to 10 years of age, with a confirmed diagnosis of peanut allergy as defined by either a positive food challenge to peanut in the past 2 years and a positive SPT or CAP-RAST to peanut, or a positive food challenge or a history of reaction to peanut ever and a positive SPT 8 mm or a CAP-RAST≥15 U/I to peanut.

[0202] Children enrolled in PPOIT-002 study were 1 to 12 years of age, with a confirmed diagnosis of peanut allergy as defined by a failed DBPCFC to peanut and a positive SPT or sIgE to peanut at screening or in the preceding 3 months.

Study Conduct

[0203] The participants of the PPOIT-001 study were randomised to either receive a peanut OIT together with the probiotic L rhamnosus, or placebo with placebo. In PPOIT-002 all participants received active treatment. The treatment began with a rush day (day 0) and the Rush dosing schedule was the same for both studies as shown in Table 3. Before initiating the Rush phase, participants received a single dose of probiotic or placebo (1 scoop dissolved into water). Thereafter, participants received increasing doses of peanut (or placebo OIT), sprinkled onto food every 30 minutes until a final dose of 12 mg peanut protein (cumulative dose 24 mg peanut protein or placebo) was reached. If a participant reacted to one of the doses during the rush phase, the rush schedule was ceased and they commenced the build-up phase at the dose immediately below the reaction-eliciting dose during the rush day, starting on the day after the rush day (i.e. day 1). The remaining rush doses that were not completed on day 0 were incorporated into the build-up phase and subsequent dose increases proceeded through all remaining doses of the rush dosing schedule followed by the doses in the build-up schedule.

[0204] Rush day was followed by the build-up phase. The two different build-up schedules for PPOIT-001 and PPOIT-002 are shown in Table 4 and Table 5, respectively. During the build-up phase, the daily dose of peanut OIT or placebo was increased every 2 weeks until a maintenance dose of 2 g was reached. Peanut protein or placebo was taken sprinkled onto food and the dose of probiotic or placebo was mixed with water and taken immediately prior to the OIT or placebo. Each dose increase was administered in hospital under medical supervision.

[0205] The dose adjustment rules were similar in both studies and included guidelines for how dosing should be continued if a participant reacted to a dose. If the reaction was mild or moderate (not anaphylaxis) and occurred within 3 days of the scheduled day of dose increase, the updosing would be deferred and the dose would be repeated before increasing, or if the reaction occurred more than 3 days prior to the scheduled day of dose increase, the updosing would proceed according to protocol. If an anaphylaxis reaction occurred, then the immediate next dose would be reduced to the previous dose amount and continued for at least 10 days before proceeding with a dose increase. If a serious anaphylaxis reaction (e.g. reduced blood pressure or loss of consciousness) occurred, then OIT would be discontinued. Time taken for each subject to reach each dose under the PPOIT-002 schedule is shown in Table 11.

[0206] Adverse events (AE) were defined as any untoward medical occurrence that occurred in a participant which did not necessarily have a relationship with the investigational product. Details regarding each AE were prospectively collected and documented on each study visit or when advised by the patient throughout the study in their study diary. The information included a description of the event, onset and stop date of the event, severity of the event, as well as suspected relationship to the study treatment. The PPOIT-001 study originally categorised all reactions that met the definition of anaphylaxis (any reaction involving airway or circulation, irrespective of how mild) as a serious adverse event (SAE). Recently, NIH/NIAID Consortium of Food Allergy Research (CoFAR) developed a standardised tool for categorisation of allergic reactions. This tool was adopted for the PPOIT-002 study and was subsequently applied to re-categorise the allergic AEs from PPOIT-001, to allow direct comparison of AE events in the two studies.

[0207] The CoFAR categorisation allocates the grading of moderate or severe to most anaphylaxis events and only those reactions that are immediately life threatening are labelled as SAE.

[0208] Briefly, the CoFAR tool grades events from 1 to 5, where grade 1 events were mild with symptoms that may include pruritus, swelling or rash or abdominal discomfort, grade 2 events were moderate with symptoms that may include persistent hives, wheezing without dyspnoea or abdominal discomfort/increased vomiting, grade 3 events were severe with symptoms that may include bronchospasm with dyspnoea, severe abdominal pain, throat tightness with hoarseness and transient hypotension, grade 4 events were life threatening and grade 5 resulted in death. Grade 4 and 5 were graded as SAE. The relationship of an AE to study product was assessed using the following categories; probably related, possibly related, unlikely to be related, unrelated.

[0209] All randomised participants were included when assessing AEs, irrespective of if and when they withdrew from the study. AEs that were categorized as unrelated to study product or unlikely to be related to study product were excluded from the present study's analyses. For the comparison between PPOIT-001 and PPOIT-002 only moderate and severe AEs and SAEs were included. Two assessors reviewed AE raw data from both PPOIT-001 and PPOIT-002 studies and applied the tool developed by CoFAR to grade severity and attribution of each event. The categorisations were then compared and if consensus reached, the categorisation was accepted. If consensus was not reached, a third independent assessor reviewed and provided final assessment.

TABLE-US-00003 TABLE 3 Peanut OIT protocol rush day (PPOIT-001 and 002) Dose Peanut protein Cumulative dose peanut protein 1 0.1 mg 0.1 mg 2 0.2 mg 0.3 mg 3 0.4 mg 0.7 mg 4 0.8 mg 1.5 mg 5 1.5 mg 3.0 mg 6 3.0 mg 6.0 mg 7 6.0 mg  12 mg 8  12 mg  24 mg

TABLE-US-00004 TABLE 4 Peanut OIT protocol build-up phase—PPOIT-001 Time following rush day at Peanut Dose which the dose commences protein  9 1 day   24 mg 10  2 weeks  25 mg 11  4 weeks  50 mg 12  6 weeks  75 mg 13  8 weeks 100 mg 14 10 weeks 125 mg 15 12 weeks 150 mg 16 14 weeks 200 mg 17 16 weeks 260 mg 18 18 weeks 330 mg 19 20 weeks 425 mg 20 22 weeks 550 mg 21 24 weeks 715 mg 22 26 weeks 925 mg 23 28 weeks 1.2 g   24 30 weeks 1.55 g   25 32 weeks 2.0 g  

TABLE-US-00005 TABLE 5 Peanut OIT protocol build-up phase—PPOIT-002 Time following rush day at Peanut Dose which the dose commences protein  9 1 day   25 mg 10  2 weeks  50 mg 11  4 weeks 100 mg 12  6 weeks 200 mg 13  8 weeks 400 mg 14 10 weeks 800 mg 15 12 weeks 1.2 g   16 14 weeks 1.6 g   17 16 weeks 2.0 g  

Calculations

[0210] Using the dates for a participant's rush day visit and subsequent build-up visits, time to reach maintenance phase and days on each dose were calculated. Time to reach maintenance was expressed as a percentage of the optimal schedule according to protocol and calculated by dividing each participant's actual time to reach maintenance phase with the optimum number of weeks it should have taken them had they progressed through the build-up protocol without deviating due to reactions or being rescheduled for other reasons. The denominator was 32 weeks for the PPOIT-001 groups and 16 weeks for PPOIT-002. The number of delayed build-up visits was extracted from the data by coding for any days on dose that was over 14 days. This would indicate a delayed build-up visit. The number of delayed build-up visits as a percentage of the total number of build-up visits was calculated by dividing the number of delayed build-up visits by the total number of build-up visits the participant had had. Delayed days were calculated by adding all days over 14 days on a dose for each participant together into a total sum of delayed days. Information on dosing on rush day, days on dose and number of missed doses was used to calculate the total cumulative dose taken during the rush day and build-up phase for each participant. To calculate the average dose taken by a participant each week of the build-up phase, the cumulative dose was divided by the number of weeks the participant was on build-up. The number of AEs, their character, severity grade, and relationship to study product was collected from the AE databases.

Statistical Analysis

[0211] Statistical analysis was done using Graphpad Prism version 7 (GraphPad Software, Inc. La Jolla, Calif., USA). The primary aims of the study were to compare time to reach maintenance, cumulative OIT dose taken during build-up, and number of moderate and severe adverse events between the three groups: PPOIT-001 active, PPOIT-001 placebo, and PPOIT-002. As an expansion of these aims, percentage of time over optimal protocol schedule, number and percentage of delayed build-up visits and total number of delayed days was compared as well. For the time to reach maintenance and delayed days analyses, only participants who reached maintenance were included, as dropouts did not have complete data for these variables. For the comparison of adverse events all participants were included irrespective of if and when they withdrew from the study. For each comparison three Mann-Whitney U test analyses were made comparing PPOIT-001 active vs PPOIT-001 placebo, PPOIT-001 active vs PPOIT-002, and PPOIT-001 placebo vs PPOIT-002. Mann-Whitney U test was chosen since the data was not normally distributed.

[0212] Between group comparisons were adjusted for four possible confounders; age, gender, diagnosis of asthma, and SPT wheal size. The subgroups are shown in Table 6. Comparisons across all three groups were performed with Kruskall-Wallis H test and two-group comparisons were performed with Mann-Whitney U test.

[0213] Univariate and multivariate regression analyses were performed to investigate the relationships between build-up duration and SU.

Ethics

[0214] PPOIT-001 and PPOIT-002 were approved by the Royal Children's Hospital Human Ethics Research Committee (HREC).

Results

[0215] In PPOIT-001, 62 children were randomized to either receive active peanut OIT with probiotic L rhamnosus (N=31) or placebo with placebo (N=31). In PPOIT-002, 20 children were assigned to receive active peanut OIT and probiotic L rhamnosus with a shortened build-up phase. The baseline demographics for the three groups are shown in Table 6. Baseline demographics between the groups were similar with the exception of asthma, age, gender and SPT wheal size. Based on these demographics all three groups were split into subgroups to confirm whether there were important differences between these subgroups (see Table 7). There were no significant differences between the subgroups.

TABLE-US-00006 TABLE 6 Demographic characteristics at study entry PPOIT 1 PPOIT 1 active placebo group group PPOIT 2 (N = 31) (N = 31) (N = 20) Age (y) Median (IQR) 6 5.6 7.8 (4.3 to 7.5) (3.4 to 8)   (5.8 to 10.73) Male sex n 17 20 14 (%) (54.8) (64.5) (70.0) History of doctor- diagnosed eczema (ever)* n 24 24 16 (%) (77.4) (77.4) (94.1) History of doctor- diagnosed asthma (ever) n 16 14 6 (%) (51.6) (45.2) (30.0) Peanut-induced SPT 18 16 11 wheal size (mm) Median (IQR) (13 to 22) (12.5 to 20)     (9 to 15.25)

TABLE-US-00007 TABLE 7 Subgroup stratifications used in adjusted analyses Age (y) Gender Asthma SPT-wheal size (mm) ≤5 Male Yes ≤10 <5 Female No <10

[0216] In the PPOIT-001 active group, 1 child withdrew during the build-up phase because of regular abdominal pain after taking the doses. In the PPOIT-001 placebo group 2 children withdrew, 1 because the family was unable to commit to the study protocol, and 1 because the mother was concerned for the safety and wellbeing of the child after an SAE. In the PPOIT-002 group 2 children withdrew, 1 because the child did not want to continue taking doses and 1 because the child had recurrent abdominal pain in association with doses after updosing to the 100 mg/day dose.

Time to Reach Maintenance was Shorter for Subjects Treated Using PPOIT-002

[0217] As shown in FIGS. 1A and 1n Table 8, the median time to reach maintenance phase was 38.4 weeks (IQR, 34.3 to 46.4) for PPOIT-001 active, 36.0 weeks (IQR, 33.4 to 37.8) for PPOIT-001 placebo, and 18.0 weeks (IQR, 16.0 to 20.0) for PPOIT-002. There was a significant difference between all three groups, PPOIT-001 active vs. PPOIT-001 placebo (P=0.03), PPOIT-001 active vs. PPOIT-002 (P<0.0001), and PPOIT-001 placebo vs. PPOIT-002 (P<0.0001).

[0218] As shown in FIG. 1B and Table 8, the median percentage of time by which a participant exceeded the optimum time to reach maintenance phase (32 weeks for PPOIT-001, 16 weeks for PPOIT-002) was 20% (IQR, 7 to 45%) for PPOIT-001 active, 13% (IQR, 4 to 18%) for PPOIT-001 placebo, and 13% (IQR, 0 to 25%) for PPOIT-002. There was a significant difference between PPOIT-001 active and PPOIT-001 placebo (P=0.03), while the values for the other comparisons were not significant, PPOIT-001 active vs. PPOIT-002 (P=0.18), PPOIT-001 placebo vs. PPOIT-002 (P=0.82).

PPOIT-002 was Associated with Fewer Delayed Build-Up Visits and Fewer Number of Delayed Updosing Days

[0219] The median number of delayed build-up visits attributable to all reasons (including reactions to treatment and logistic reasons) was 5 (IQR, 3 to 7) for PPOIT-001 active, 4 (IQR, 3 to 6) for PPOIT-001 placebo, and 1 (IQR, 0 to 2) for PPOIT-002. There was a significant difference between PPOIT-001 active and PPOIT-002 (P<0.0001), and between PPOIT-001 placebo and PPOIT-002 (P<0.0001).

[0220] For PPOIT-002, the median number of build-up visits that were delayed due to treatment related reactions was 0 (IQR, 0 to 1). The median percentage of build-up visits that were delayed due to treatment was 0% (IQR, 0 to 13%).

[0221] The median percentage of build-up visits that were delayed was 31% (IQR, 19 to 44%) for PPOIT-001 active, 25% (IQR, 19 to 38%) for PPOIT-001 placebo, and 13% (IQR, 0 to 22%) for PPOIT-002. There was a significant difference between PPOIT-001 active and PPOIT-002 (P<0.0001), and PPOIT-001 placebo and PPOIT-002 (P=0.002).

[0222] The median total number of days a participant exceeded their dosing protocol (e.g. stayed on a dose for longer than 14 days) was 48 days (IQR, 18.5 to 87) for PPOIT-001 active, 29 days (IQR, 13 to 50) for PPOIT-001 placebo, and 14 days (IQR, 0 to 19.5) for PPOIT-002. There was a significant difference between all three groups, PPOIT-001 active vs. PPOIT-001 placebo (P=0.04), PPOIT-001 active vs. PPOIT-002 (P<0.0001), and PPOIT-001 placebo vs. PPOIT-002 (P=0.001).

[0223] The results are summarised in FIGS. 2A, B and C, and percentage calculations are provided in Table 8.

Cumulative Dose Taken During Build-Up

[0224] The median cumulative dose taken during build-up phase was 108.50 g (IQR, 95.44 to 126.20) for PPOIT-001 active, 97.49 g (IQR, 92.64 to 108.60) for PPOIT-001 placebo, and 61.31 g (IQR, 59.40 to 62.34) for PPOIT-002. There was a significant difference between all three groups, PPOIT-001 active vs. PPOIT-001 placebo (P=0.02), PPOIT-001 active vs. PPOIT-002 (P<0.0001), and PPOIT-001 placebo vs. PPOIT-002 (P<0.0001).

[0225] The median average dose taken per week in the build-up phase was 2.77 g (IQR, 2.46 to 3.12) for PPOIT-001 active, 2.89 g (IQR, 2.65 to 2.96) for PPOIT-001 placebo, and 3.45 g (IQR, 3.11 to 3.65) for PPOIT-002. There was a significant difference between PPOIT-001 active vs PPOIT-002 (P<0.0001), and PPOIT-001 placebo vs. PPOIT-002 (P<0.0001).

[0226] The results are summarised in FIGS. 3A and B and Table 8.

PPOIT-002 had Fewer Moderate or Severe AEs During Build-Up Phase Compared to PPOIT-001

[0227] Data on moderate and severe AEs during build-up phase were available for both PPOIT-001 and PPOIT-002 and were compared. AEs determined to be unlikely or unrelated to study product were excluded. As shown in FIG. 4 and

[0228] Table 9, at least 1 moderate or severe AE was reported in 35.5% of children in the PPOIT-001 active group, 19.4% in the PPOIT-001 placebo group, and 25.0% in the PPOIT-002 group. The total number of moderate and severe AEs were 20 in the PPOIT-001 active group, 12 in the PPOIT-001 placebo group, and 6 in the PPOIT-002 group. The number of moderate and severe AEs per participant did not differ significantly by group, median 1.5 (IQR, 0-1) for the PPOIT-001 active group, 0 (IQR, 0-0) for the PPOIT-001 placebo group, and 0 (IQR, 0-0.75) for the PPOIT-002 group. During build-up a total of 0.23% (20/8577) of doses resulted in a moderate or severe reaction in the PPOIT-001 active group, 0.16% (12/7364) in the PPOIT-001 placebo group, and 0.24% (6/2449) in the PPOIT-002 group.

[0229] During the build-up phase, there was one SAE recorded that was related to study product in the PPOIT-001 placebo group and was an episode of severe abdominal pain and throat tightness. There were no SAE during build-up phase for active PPOIT-001 group or the PPOIT-002 group.

[0230] For the PPOIT-002 group, 3 out of the moderate and severe AEs during build-up phase occurred on a build-up day and 3 occurred during home dosing. All of the moderate and severe AEs in the PPOIT-002 group occurred during the first three weeks of the build-up phase (data not shown).

[0231] Table 10 shows the frequency of AEs for PPOIT-001 and PPOIT-002 and Table 11 shows the time taken for each subject to reach each dose under the PPOIT-002 schedule.

TABLE-US-00008 TABLE 8 Time to reach maintenance, cumulative dose peanut or placebo taken during build-up phase, and delayed build-up visits and days. Kruskall-Wallis test performed to determine p-values PPOIT 1 PPOIT 1 PPOIT active placebo 2 P- (N = 30) (N = 29) (N = 18) value Time to reach 38.4 36 18 <0.0001 maintenance (weeks) Median (IQR) (34.3 to (33.4 to (16 to 46.4) 37.8) 20) Percentage 20 13 13 0.0886 of time over protocol Median (IQR) (7 to 45) (4 to 18) (0 to 25) Cumulative dose 108.5 97.49 61.31 <0.0001 taken during build-up (g) Median (IQR) (95.44 to (92.64 to (59.40 to 126.2) 108.6) 62.34) Average 2.77 2.89 3.45 <0.0001 dose taken each week (g) Median (IQR) (2.46 to (2.65 to (3.11 to 3.12) 2.96) 3.65) Delayed days 48 29 14 <0.0001 on dose Median (IQR) (18.5 to (13 to (0 to 87) 50) 19.5) No. of delayed 5 4 1 <0.0001 build-up visits Median (IQR) (3 to (3 to 6) (0 to 2) 7.25) Percentage of 31 25 13 0.0003 delayed build- up visits for any reason Median (IQR) (19 to 44) (19 to 38) (0 to 22) No. of build- N/A N/A 0 up visits delayed due to treatment related reactions Median (IQR) (0 to 1) Percentage of N/A N/A 0 build-up visits delayed due to treatment related reactions Median (IQR) (0 to 13)

TABLE-US-00009 TABLE 9 Characteristics of severe and moderate AEs PPOIT 1 PPOIT 1 active placebo group group PPOIT 2 (N = 31) (N = 31) (N = 20) Patients who n 11   6   5 experienced (%) (35.5) (19.4) (25) ≥1 AE No. of AEs per patient 0 n 20   25   15 (%) (64.5) (80.6) (75) 1 n 7  3   4 (%) (22.6)  (9.7) (20) 2 n 1  1   1 (%)  (3.2)  (3.2)  (5) 3 n 1  1   0 (%)  (3.2)  (3.2)  (0) 4 n 2  1   0 (%)  (6.5)  (3.2)  (0) Total no. n 20   12    6 of AEs Median 0  0   0 (IQR) (0 to 1) (0 to 0) (0 to 0.75) No. of AEs by time point Modified n 1  1   1 rush day (%) (5)   (8.3)  (16.7)  Build-up n 19   11    5 phase (%) (95)   (91.7)  (83.3)  Home n N/A N/A  3 dosing (%) (50) Updosing n N/A N/A  3 day (%) (50)

TABLE-US-00010 TABLE 10 Frequency of AEs as a percentage of total doses PPOIT 1 active PPOIT 1 placebo PPOIT 2 group (N = 31) group (N = 31) (N = 20) Moderate % 0.22 0.15 0.20 AEs (n) (19 of 8577) (11 of 7364) (5 of 2449) Severe % 0.01 0.01 0.04 AEs (n)  (1 of 8577)  (1 of 7364) (1 of 2449) SAEs % 0 0.01 0 (n)  (1 of 7364)

TABLE-US-00011 TABLE 11 Time taken for each subject to reach each dose under the PPOIT2 schedule. days to days to days to days to days to days to days to days to days Patient no. 25 mg 50 mg 100 mg 200 mg 400 mg 800 mg 1200 mg 1600 mg to 2 g 01 1 14 28 42 57 70 84 98 112 02 1 14 28 42 63 77 91 105 119 03 1 14 28 42 56 70 85 98 112 04 1 14 28 42 56 70 84 98 112 05 1 14 33 51 60 75 89 103 117  06.sup.a 91 105 126 140 154 168 182 196 210 07 1 14 30 44 72 98 112 126 140 09 1 14 28 42 56 70 84 98 112  10.sup.b 1 70 84 98 136 140 161 175 203  12.sup.c 1 42 56 70 84 98 112 126 140 13 1 14 28 56 70 84 98 112 126 14 1 14 28 42 56 70 84 98 112 16 1 19 33 61 75 89 103 117 131  17.sup.d 42 56 70 84 98 112 126 140 189 18 1 28 56 70 84 98 112 126 140 19 1 28 42 56 70 84 98 112 126 Summary statistics mean 9.1875 29.625 45.375 61.375 77.9375 88.86 103.59 117.82 134.36 median 1 14 31.5 53.5 70 84 98 112 126 IQR 1-1   14-31.5 28-56 42-70 56.75-84   70-98 84.75-112    98-126 112-140 tests for normality (performed on SPSS) Kolmogorov- 0.026 0.016 0.017 0.001 Smirnov Shapiro-Wilk 0.002 0.001 0.001 0.001 Footnote to Table 11: .sup.areacted to 0.8 mg on RUSH day .sup.breacted during BU to 50mg .sup.creacted during BU to 50mg .sup.dreacted to 1.5 mg on RUSH day

Likelihood of Achieving Sustained Unresponsiveness at the End-of-Treatment.

[0232] At the end of 18 months of treatment for both PPOIT 001 and PPOIT 002, the presence of sustained unresponsiveness (SU) was tested by double blind placebo controlled peanut challenge.

[0233] Using pooled data from PPOIT-001 active group and POIT-002, a univariate regression analysis was performed to determine whether the duration of the build-up phase was associated with the likelihood of achieving sustained unresponsiveness at the end-of-treatment. We found that a shorter build-up phase was associated with a significantly increased likelihood of achieving sustained unresponsiveness. Specifically, for every week that the build-up phase is longer, the likelihood or more specifically the odds of a subject achieving sustained unresponsiveness was 7.5% (1-0.925) lower, p=0.026 (see Table 12). Moreover, the median time in build-up phase was 11 weeks shorter in those who achieved sustained unresponsiveness compared to those who did not. In contrast, the total cumulative dose of peanut protein received during OIT and the dose reached on rush day were not associated with likelihood or odds of achieving sustained unresponsiveness. Univariate regression analyses showed that asthma at study entry and allergic rhinitis at study entry also influenced the likelihood or odds of achieving SU. A logistic regression model was therefore run to estimate the effects of asthma, allergic rhinitis and build-up duration on SU. This revealed that Build-up duration remained a significant predictor of SU. Specifically, in this model including both baseline asthma and allergic rhinitis, the likelihood or odds of achieving SU were 1.1 time greater for every 1 week reduction in build-up duration.

TABLE-US-00012 TABLE 12 Odds Correlation between build- Ratio of up time and sustained achieving p 95% unresponsiveness SU value CI For every 1 week that Build-up 0.925 0.015 0.87- time is increased (unadjusted 0.99 univariate regression analysis) For every 1 week that Build-up time 0.91 0.025 0.84- is increased (Logistic regression 0.99 model including baseline asthma and allergic rhinitis)

Discussion

[0234] The purpose of this study was to compare the time to maintenance dose in PPOIT-001 (32 weeks) with that of PPOIT-002 (16 weeks). Specifically, the time to reach maintenance, delays in scheduled build-ups, cumulative dose taken during

[0235] Rush and build-up phase, and number of adverse events were compared. This study found that the PPOIT-002 group's build-up phase was either equal or superior to both PPOIT-001 active and PPOIT-001 placebo build-up phase in terms of tolerability and frequency of adverse events.

[0236] The time to reach maintenance was significantly shorter for the PPOIT-002 group. This is to be expected because the schedule was half as long as PPOIT-001. In addition, the PPOIT-002 build-up schedule was well tolerated despite being shorter. Had the new build-up schedule not been well tolerated, then delays because of reactions would have put the median time to reach maintenance for the PPOIT-002 group (which was 18 weeks) closer to the PPOIT-001 active (38 weeks) and PPOIT-001 placebo (36 weeks) groups. When adjusting for the different build-up schedules by only looking at the median percentage of time that the participants exceeded the optimum time to reach maintenance, one can still see that PPOIT-002 (13% over optimum time) is equal to the PPOIT-001 placebo group (13%) and superior to the PPOIT-001 active group (20%).

[0237] When comparing protocol deviations in the form of delayed build-up visits and number of delayed days FIG. 2 shows that the PPOIT-002 group deviated significantly less from the build-up protocol than both PPOIT-001 active and PPOIT-001 placebo. When only looking at the PPOIT-002 group it is clear that the median number of build-up visits that were delayed because of treatment related reactions is 0, meaning that the majority of build-up visits were delayed due to logistic reasons (e.g. public holidays, family being unable to attend, study team unable to accommodate scheduled visit) and the more rapid dose escalation protocol of PPOIT-002 was not associated with an increased number of adverse reactions that resulted in delayed build-up visits. A longer build-up phase equals more build-up visits that could in theory be delayed because of logistic reasons, which could be one of the reasons why the PPOIT-001 participants deviated more from protocol since they had twice as many build-up visits to attend. The very few number of build-up visits in PPOIT-002 that were delayed because of treatment show that the build-up was well tolerated and did not often lead to complications in the form of reactions that would force a protocol deviation and delay.

[0238] The number of AEs were lower in the PPOIT-002 group compared with the PPOIT-001 active group, although there was overall a very small number of AE that occurred: 20 (0.21% of doses given) in PPOIT-001 active, 12 (0.16% of doses given) for PPOIT-001 placebo, and 6 (0.24% of doses given) for PPOIT-002. At the very least, PPOIT-002 was non-inferior compared to PPOIT-001 in terms of safety and tolerability despite incorporating a more rapid dose escalation regimen. The cumulative dose taken during build-up phase is naturally smaller for PPOIT-002 as they were on build-up for half the time that subjects in PPOIT-001 active were. When looking at the average dose taken each week, PPOIT-002's is higher since the updosing was steeper with doses being doubled up until the 800 mg dose. When calculating what the optimum dose taken for the entire treatment course is (assuming a participant follows the dosing protocol and does not miss any doses) then the total cumulative dose would be 654 g for PPOIT-001 (32 weeks build-up and 40 weeks maintenance), and 845 g for PPOIT-002 (16 weeks build-up and 56 weeks maintenance).

[0239] The multi-dimensional approach provided herein provides more comprehensive understanding of the tolerability and safety of the new dose escalation schedule for PPOIT-002 when compared to PPOIT-001. Time to reach maintenance can be used to assess the tolerability of the treatment together with the percentage of time over optimum, and number of delayed build-up visits. Had the new dose escalation schedule been poorly tolerated then the time to reach maintenance would have been longer, the percentage of time over optimum would be higher, and there would have been more delayed build-up visits.

[0240] When comparing the moderate to severe AEs for PPOIT-001 and PPOIT-002, it was unexpectedly observed that PPOIT-002 had substantially similar rates of moderate to severe AEs despite the fact that the build-up phase in PPOIT-002 was half the period of time. The rates of AEs from PPOIT-001 and PPOIT-002 are only for the rush and build-up phases of the treatment and may therefore be lower than what they would have been had AEs from the entire treatment span been included (i.e. rush, build-up and maintenance phase). However, most peanut OIT studies that have stratified AE rates based on treatment phase, have found that the majority of AEs occur in the beginning of treatment, during the rush day and build-up phase and therefore it is unlikely that the AE rates would be significantly higher than shown here.

[0241] Compared to many of these studies the AE reporting in PPOIT-002 and PPOIT-001, is robust and gives an accurate image of the AEs that occurred during the studies being based on the CoFAR severity grading scale.

[0242] Previous peanut OIT schedules have applied gradual dose escalation to avoid AEs as most reactions occur during the build-up phase particularly on days when the dose is increased. Therefore, the prior art has shifted to slower dosing schedules in order to minimise the rate of AEs during dose escalation—for example a recent large multicentre trial of 300 mg OIT applied a build-up phase of 26 weeks to reach 300 mg (PALISADE study—Vickery 2018 NEJM, supra.). The improved safety profile of PPOIT-002 is believed to be due to the faster build-up schedule of peanut OIT since that was the only parameter of dosing that was modified. While it is possible that the probiotic enabled the faster build-up schedule to be successfully applied, it is apparent that the only change between PPOIT-001 and PPOIT-002 was the faster build-up schedule (completed in half the time compared with the PPOIT-001 schedule). For peanut OIT to be implemented in a clinical setting it has to be practical. A long build-up phase where participants have to visit the hospital every two weeks for updosing is both demanding on families and resource intensive since there has to be a doctor and nurse present for each visit. A shorter build-up with steeper dose increases means fewer visits and less resources spent, something that has until now not proven feasible because of a higher rate of AEs and less tolerability. However, the findings in this study surprisingly suggest that using a faster build-up schedule may be possible without compromising safety. Furthermore, the shorter build-up was unexpectedly associated with a higher likelihood or odds of achieving sustained unresponsiveness. The comparison done in our study between PPOIT-001 and PPOIT-002, where the only thing that was changed was the dose escalation schedule, indicates that a shorter dose escalation schedule is possible for treating peanut allergy without reducing tolerability and compromising safety.

Example 2: Determination of Induction of pDCS by Probiotics

[0243] This example reports the results of experiments in which the effects of probiotics on the number of circulating plasmacytoid dendritic cells (pDC) after oral administration of the probiotic for 2 weeks in healthy adults with or without allergic disease were examined.

Materials and Methods

Administration of Probiotics

[0244] 120 healthy adults were recruited. Six different commercially available probiotics were tested (Table 13). Each probiotic was administered to 20 healthy adults as a daily standard dose for 2 weeks. Participants were assigned sequentially to receive one of the six probiotics. Subjects were considered ‘healthy’ in that they were generally well without serious illness and not taking immunomodulatory therapies. Presence of allergic disease was not an exclusion criteria, as 40%-50% of the Australian population are affected by one or more of the allergic conditions (asthma, allergic rhinitis, eczema, food allergy). To determine if the effects of probiotics were similar in adults with and without allergic disease, subjects were subdivided into those without allergic disease and those with allergic disease. Details of the allergic diseases present in the healthy adults is shown in Table 14.

TABLE-US-00013 TABLE 13 Probiotic dosages Probiotic Dose Lactobacillus rhamnosus GG 20 billion CFU/day Bifidobacterium lactis 10 billion CFU/day Yeast (Saccharomyces boulardii) 10 billion CFU/day E. coli Nissle 1917  1 billion CFU/day Streptococcus thermophilus 20 billion CFU/day Bifidobacterium breve 20 billion CFU/day

TABLE-US-00014 TABLE 14 Description of allergic disease amongst tested subjects. E. coli All B. S. Nissle S. subjects LGG lactis boulardii 1917 thermophilus B. breve N = 120 N = 20 N = 20 N = 20 N = 20 N = 20 N = 20 Age years 35.5  32.5  37.5  34.0  39.5  38.0  31.0  (SD) median (9.8) (9.6) (11.0) (7.8) (8.8) (9.0) (12.2) Female 95   17   17   18   5   4   0   sex n (%) (79) (85) (85) (90) (75) (80) (0) Food 2   0   2   0   0   0   0   allergy (2) (0) (10) (0) (0) (0) (0) n (%) Asthma 25   2   3   6   3   5   6   n (%) (21) (10) (15) (30) (15) (25) (30) Eczema 17   3   6   4   3   1   0   n (%) (14) (15)  (30) (20) (15) (5) (0) Allergic 38   6   8   7   7   5   5   rhinitis (32) (30)  (40) (35) (35) (25) (26.3) n (%) Any 58   9   12   13   8   7   9   allergic (48) (45) (60) (65) (40) (35) (45) disease n (%) No allergic 62   11   8   7   12   13   11   disease (52) (55) (40) (35) (60) (65) (55) n (%)

Blood Collection

[0245] A 20 mL blood sample was collected prior to probiotic administration (day 0) and after 14 days of probiotic administration (day 14). Blood samples were processed by density gradient centrifugation within 2 hours of collection to separate the plasma and peripheral blood mononuclear cells (PBMCs). PBMCs were cryopreserved and stored in liquid nitrogen for future batched analyses.

Cell Culture for 48 hrs

[0246] PBMCs were thawed using the ThawSTAR Automated Cell Thawing System (MedCision, San Rafael, Calif.). Upon thawing, cells were added dropwise to 10 ml of cold RPMI (Gibco Life Technologies, Grand Island, N.Y.) with 2% Fetal Bovine Serum (FBS) and spun at 400 g for 5 mins. Supernatant was removed and cells were resuspended in 1 ml of 2% FBS in RPMI in preparation for counting. Cell counts were determined by trypan blue exclusion using TC20 automated cell counter (Biorad, Hercules, Calif.).

[0247] PBMC cultures were set up in round bottom 96-well plate with 200,000 cells/well incubated with or without antigen (heat-killed probiotic or media alone) in AIM-V serum free medium (Gibco Life Technologies, Grand Island, N.Y.) with 4×10.sup.−5 M 2-mercaptoethanol for 48 hrs at 37° C. in 5% CO2.

Flow Cytometric Analysis of pDC

[0248] Prior to analyses, plates were spun at 400 g, 4° C. Supernatant was removed and placed in another 96-well plate and frozen at −80° C. for future analysis. Cells remained in the wells they were cultured in, 200 μl of PBS was added to each well and plates spun at 400 g, 4° C. Plates were ‘flicked’ into the sink to remove PBS wash. One hundred microliters of Fixable Viability Dye 510 (BD Biosciences, San Jose, Calif.) prepared at 0.5 μl/ml in PBS was added to each well, mixed thoroughly and incubated at room temperature (RT), light protected for 15 mins. One hundred microliters of FACS buffer (2% FBS in PBS) was added to each well and plates spun at 400 g, 4° C. and flicked. Antibody cocktail consisting of anti-human HLA DR BB515, CD123 PerCPy5.5, CD11c PE-Cy7, CD1c-PE, lineage (CD3, CD19, CD20 APC-H7 and CD56) APC-Cy7, CD14 V450 and CD16 BUV395 (all antibodies purchased from BD Biosciences, San Jose, Calif. except for CD123 and CD56 which were from Biolegend, San Diego, Calif.) were added to each well at a predetermined titre (Table 15) in 40 μl of FACS buffer, mixed thoroughly and incubated for 20 mins on ice. Unbound antibody was washed out in 200 μl of FACS buffer, plates spun and flicked, then 150 μl of FACS buffer added to each well for analysis on the Fortessa X-20 high throughput system (HTS) (BD Biosciences, San Jose, Calif.) Compensation controls for each fluorophore were prepared on anti-mouse Ig CompBeads (BD Biosciences, San Jose, Calif.) with compensation performed by BD FACSDIVA software (BD Biosciences, San Jose, Calif.). About 200,000 events were collected per well.

TABLE-US-00015 TABLE 15 Antibody titres, fluorophore, clone, vendor and catalogue number. Antibody Fluorophore Dilution Clone Vendor Cat# HLA DR BB515 1:200 G46-6 BD 564516  CD123 PerCPCy5.5 1:20  7G3 BD 558714  CD11c PE-Cy7 1:100 B-Ly6 BD 561356 CD1c PE 1:40  F10/21A3 BD 564900 Lineage (CD3, APC-H7 BD CD19, CD20, CD56) CD3  APC-H7 1:100 SK7 BD 560176 CD19 APC-H7 1:100 SJ25C1 BD 560177 CD20 APC-H7 1:100 2H7 BD 560734 CD56  APC-Cy7 1:100 NCAM Biolegend 362512 CD14 V450 1:200 MOP9 BD 560349 CD16 BUV395 1:200 3G8 BD 563785 Live/dead BV510 564406 BV510

TABLE-US-00016 Fortessa X-20 HTS settings Sample Flow Rate (μl/sec) 2.5 Sample Volume (μl) 125 Mixing Volume (μl) 100 Mixing Speed (μl/sec) 200 Number of Mixes 3 Wash Volume (μl) 400

Post-Acquisition Analysis

[0249] Post-acquisition analysis was performed by FlowJo v10.3 (FlowJo, LLC, Ashland, Oreg.). pDC were identified as HLA-DR+Lineage-(CD3, CD19, CD20, CD56) CD14−CD16−CD11c−CD123+ cells. The number of pDC in HK probiotic stimulated cultures was calculated by subtracting the proportion of pDC in unstimulated cultures from the proportion of pDC in HK probiotic stimulated cultures to adjust for nonspecific variation in baseline pDC numbers between subjects and across different time points.

Statistical Analysis

[0250] Data are presented as the median and interquartile range for each time point.

Results

Effect of In Vivo Probiotic Supplementation on pDC Proportions in Peripheral Blood

[0251] Median proportions of pDC in PBMC cultures from healthy adults were increased in all probiotic groups at day 14 (14 days after treatment) compared with day 0 (Table 16). pDC proportions increased by 2-fold from day 1 to day 14 with LGG (from 8.49 to 17.56%), B. lactis (increase from 6.31 to 11.54), S. boulardii (from 1.85 to 3.12%) and E. coli Nissle 1917 (from 3.76 to 7.21%) and increased by 4 fold from day 1 to day 14 with S. thermophilus (from 0.45 to 1.91) and B. breve (from 0.61 to 2.45).

[0252] The effects of probiotics on pDC proportions were then examined according to presence or absence of allergic disease to determine if probiotic effects were similar for healthy adults with and without allergic disease. At both day 0 (before probiotic treatment) and at day 14 (after probiotic treatment), the proportions of pDCs in adults with and without allergic disease were similar (and not significantly different, p>0.05), indicating that probiotic induction of pDCs is not altered by the presence of allergic disease (FIG. 5).

TABLE-US-00017 TABLE 16 pDC proportions in PBMC cultures from healthy adults treated with probiotic for 14 days Pre- Post- treat- treat- ment IQR ment IQR (day 0) 25-75% (day 14) 25-75% Fold- Probiotic median percentile median percentile change LGG 8.49  2.61-13.08 17.56 6.10-23.78 2.1 B. lactis 6.31  2.39-13.26 11.54 4.61-23.75 1.8 S. boulardii 1.85 0.48-2.53 3.12 0.97-5.06  1.7 E. coli Nissle 3.76 2.40-6.52 7.21 4.63-13.97 1.9 1917 S. thermophilus 0.45 −2.02-2.22   1.91 −1.93-7.36    4.2 B. breve 0.61 −0.26-3.19   2.45 1.1-7.12 4.0

[0253] The data show that oral administration of six different probiotics from different genera to healthy adults for 14 days leads to systemically detectable increases in pDC proportions. The effect of oral probiotic administration on the pDC response capacity was assessed by measuring the number of pDC generated in vitro (in HK probiotic stimulated cultures to mimic the in vivo exposure) before and after the oral administration of probiotic. The data show that the production of pDC is greater after 14 days of probiotic supplementation compared with day 0. The increases in pDC numbers following 14 days of oral administration of probiotic are consistent with an increased capacity to induce antigen-specific Treg which in turn inhibit the allergic response (Colonna et al, Nat Immunol 5:1219-1226, 2004; Akdis and Akdis, WAO J. 8:17, 2015). pDC present encountered antigens to antigen-specific naïve T cells and direct the naïve T cells to differentiate into regulatory T cells (Treg). The effects of probiotics on pDCs was similar in adults with and without allergic disease (FIG. 5).

Example 3—Comparison of PPOIT-003 Data to Prior Art

[0254] PPOIT-003 was a 3 arm multicentre randomised controlled clinical trial conducted using the same build-up protocol as PPOIT-002. Subjects were randomised to receive one of three interventions—placebo, probiotic added to peanut OIT, and peanut OIT without probiotic. The treatment period was 18 months, after which patients were tested for both desensitization and sustained unresponsiveness (SU), using the same food challenge as used in PPOIT2.

Comparison of SU Rates Achieved Following Peanut OIT (without Probiotic) Using the Shorter Build-Up Regimen with the Prior Art (Vickery 2014 Supra.)

[0255] A randomised trial was conducted comparing probiotic peanut OIT vs peanut OIT alone vs placebo (PPOIT-003 study). OIT alone induced SU in 50.6% of subjects compared with 5.1% of placebo treated subjects. This rate of SU with peanut OIT is higher than what has been reported when OIT was administered using a longer duration in a similar population of subjects with similar age and peanut SPT wheal size (Table 17). This suggests that peanut OIT administered using a shorter duration led to a higher rate of SU compared with peanut OIT administered using a longer duration. Furthermore, the prior study of peanut OIT using a longer duration also used a higher maintenance dose and shorter period of allergen elimination before testing for SU, which are both considered to increase the likelihood or odds of achieving SU.

TABLE-US-00018 TABLE 17 Comparison of OIT arm in PPOIT-003 study with OIT Prior art PPOIT- PPOIT- Vickery 003 study 003 study 2014 supra OIT Placebo OIT (n = 83) (n = 39) (n = 39) Build-up 16 weeks to 16 weeks to 20 weeks to 300 mg duration 2000 mg 2000 mg followed by 4-5 years to 4000 mg SU 50.6% 5.1% 30.8% (ITT) (42/83) (2/39) (12/39) SU measure 8-week SU 8-week SU 4-week SU 5000 mg 5000 mg 4000 mg OIT maintenance 2000 mg 2000 mg 4000 mg dose Age range 1-10 years 1-10 years 1-16 years (median) (5.7 years) (5.9 years) Peanut SPT mm 12.6 12.7 Approx. 12.5 Mean (SD) (5.20) (5.82)

[0256] Previous studies have suggested that longer total duration of treatment and higher maintenance OIT dose allow an increase in the sustained unresponsive (SU) rate. Therefore, the higher SU rate in a similar age group of patients demonstrated with peanut OIT in the PPOIT-003 study, despite shorter overall duration of the treatment and lower maintenance dose was surprising. It is postulated this surprising result is due to the more rapid initial dosing to achieve 2000 mg in as little as 16 weeks.

Comparison of Safety and Tolerability of Peanut OIT Using a Shorter Build-Up Duration with the Prior Art

TABLE-US-00019 TABLE 18 Comparison of safety events in the OIT arm of the PPOIT-003 study with the OIT arm of the PALISADE study (Vickery 2018 NEJM, supra.) PPOIT-003 PALISADE Study Study OIT Placebo AR101 Placebo n = 83 n = 39 n = 372 n = 124 Gastrointestinal Abdominal 54.2% 33.3% 52.2% 24.2% pain (45) (13) (194) (30) Vomiting 31.3% 17.9% 41.4% 24.2% (26)  (7) (154) (30) Respiratory Cough 22.9% 0% 40.9% 33.9% (19)  (0) (152) (42) Dyspnea  1.2% 5.1% 11.8%  4.0%  (1)  (2)  (44)  (5) Multiple 10.8%  5.1% Not Not respiratory  (9)  (2) reported reported symptoms Skin and subcutaneous Urticaria 54.2% 46.2% 38.4% 24.2% (45) (18) (143) (30) Serious  4.8%  0%   5.6%  1.6% Adverse Events (SAEs) Overall  20.5%* 10.3% 21.0%  7.3% withdrawal rate Withdrawals  3.6%  0%  11.6%  2.4% due to AE’s *PPOIT-003 OIT withdrawal rate includes 4 subjects who withdrew from treatment but remained on study. The rate of study withdrawals was 15.7% (13/83).

[0257] The greatly increased peanut OIT dosing schedule in PPOIT-003 (2000 mg reached within 16 weeks compared with 300 mg reached in 24 weeks in PALISADE) would have been expected to result in an increased number of adverse events. We have compared—Proportion of subjects reporting TEAEs involving GI, Respiratory and Skin (SOC and PT), as well as total SAEs, overall withdrawal rates and withdrawals due to AEs. It is surprising that even without the probiotic these events are not increased.

[0258] The exposure adjusted incidence rate of AEs can also be used to compare safety of the shortened schedule with slower schedules. Exposure adjusted incidence rate of AEs is calculated by dividing the number of AEs by the actual years of treatment, allowing direct comparison of treatments where there is variation in duration of treatment. Table 19 compares the exposure adjusted incidence of AEs reported with peanut OIT (2000 mg maintenance dose) administered using a shorter duration (in the PPOIT-003 study) to peanut OIT administered using a longer and lower 300 mg maintenance dose (in the PALISADE study). The exposure adjusted incidence rate of AEs is approximately 4 times lower with the faster schedule despite reaching a 6-7 fold higher maintenance dose.

TABLE-US-00020 TABLE 19 Comparison of exposure adjusted incidence rate of AEs in the OIT arm of the PPOIT-003 study with the OIT arm of the PALISADE clinical trial (Vickery 2018 NEJM, supra). PPOIT-003 trial PALISADE trial 2000 mg maintenance dose 300 mg maintenance dose Years of Years of Number Exposure Exposure Number Exposure Exposure of (on adjusted of (on adjusted TEAEs treatment) rate TEAEs treatment) rate OIT 1228 108.0 11.4 12489 307.0 40.7 Placebo 116 55.5 2.1 617 108.8 5.7

Comparison of the Effect of Inclusion of the Probiotic on Safety Signals

[0259] In the PPOIT003 study, a direct comparison of the effect of adding the probiotic to peanut OIT can be made by comparing subjects receiving probiotic peanut OIT vs those receiving peanut OIT alone. The rate of withdrawals, dose adjustments during build-up phase, proportion of subjects reporting AEs, exposure adjusted incidence rate of AEs can provide information on the tolerability and safety of the treatment schedule.

[0260] All oral immunotherapy protocols allow for dose adjustments where a child has a severe reaction to the dose being given. In these circumstances the dose is reduced for a period of time. As a result, the number of dose adjustments occurring during the escalation phase gives an indication of how well tolerated the treatment was in that cohort.

TABLE-US-00021 TABLE 20 Comparison of dose adjustments in the PPOIT-003 study in subjects receiving probiotic with peanut OIT (PPOIT) and peanut OIT alone (OIT) PPOIT OIT Placebo (n = 79) (n = 83) (n = 39) Subjects requiring dose 10.1% (8)  16.9% (14) 12.8% (5) adjustment during build-up Total number of dose 12 16 8 adjustments Dose at which adjustment occurred, % (n)  3 mg-400 mg 16.7% (2)  56.3% (9)  75.0% (6) 800 mg-2000 mg 83.3% (10) 43.8% (7)  25.0% (2)
The number of withdrawals from a study or from treatment provides an indication of tolerability of a treatment. In the PPOIT-003 study, there were fewer withdrawals in subjects receiving Probiotic together with OIT compared with subjects receiving OIT alone (Table 21)

TABLE-US-00022 TABLE 21 Comparison of withdrawals in the PPOIT003 study in subjects receiving peanut OIT with probiotic (PPOIT) and peanut OIT alone (OIT) PPOIT OIT Placebo (n = 79) (n = 83) (n = 39) Withdrawals* 12.7% (10) 20.5% (17) 10.3% (4) Withdrawals due to AE 2.6% (2) 3.6% (3)  0.0% (0) *Includes 6 subjects who withdrew from treatment but remained on study—2 in the PPOIT group and 4 in OIT group.

Effects in 1-5 Year Olds

[0261] The beneficial effect of the probiotic was particularly pronounced in children between the ages of 1 and 5 years as shown in Tables 22 & 23 which show a comparison of the withdrawal rates and adverse events in the 1 to 5 year age group in subjects treated with probiotic+OIT (PPOIT) and OIT alone. Results show that in this younger age group, the probiotic improves tolerability of the peanut OIT treatment (fewer withdrawals), likely due to a protective effect against systemic and gastrointestinal side effects.

TABLE-US-00023 TABLE 22 Comparison of withdrawals in the 1-5 year old age group PPOIT OIT Placebo Reason for withdrawal n = 41 n = 43 n = 20 Total 4.9% (2/41) 18.6% (8/43) 10.0% (2/20) Adverse event 0.0 (0) 2.4 (1) 0.0 (0) Lack of patient compliance 2.4 (1) 4.7 (2) 0.0 (0) Patient/Caregivers decision 2.4 (1) 7.0 (3) 5.0 (1) Relocated while in the study 0.0 (0) 0.0 (0) 5.0 (1) Study is an inconvenience 0.0 (0) 0.0 (0) 0.0 (0) Other (specify) 0.0 (0) 4.7 (2) 0.0 (0)

TABLE-US-00024 TABLE 23 Comparison of adverse events in the 1-5 year old age group PPOIT OIT Effect size Children aged 1-5 years N = 41 N = 43 (Delta) Related Moderate/Severe AEs Number of subjects 10 20 22% reduction (24.4%) (46.5%) Number of events 27 43 37% reduction Exposure adjusted 0.439 0.667 0.228 incidence* reduction Related Moderate/Severe Systemic Reactions Number of subjects 9 17 17.5% (22%) (39.5%) reduction Number of events 16 30 46.7% reduction Exposure adjusted 0.260 0.465 0.205 incidence* reduction GI Disorders (All TEAEs) Number of subjects 29 29 3.3% increase (70.7%) (67.4%) Number of events 156 214 27% reduction Exposure adjusted 2.537 3.318 0.781 incidence* reduction Immune disorders (All TEAEs) Number of subjects 23 28 9% reduction (56.1%) (65.1%) Number of events 97 161 40% reduction Exposure adjusted 1.577 2.496 0.919 incidence* reduction

Example 4—Effect of PPOIT and OIT on Peanut sIgE Levels

[0262] Peanut sIgE is responsible for the presence of allergy to peanut and initiates the cascade of immune changes that lead to symptoms of an allergic reaction. Peanut sIgE provides a biomarker for clinical peanut allergy. The absence of peanut sIgE, or a negative test results for peanut sIgE<0.35 kU/L, is a highly specific marker for absence of IgE mediated allergy to peanut. Conversely, although presence of peanut sIgE on its own does not necessarily result in clinical peanut allergy, the higher the level of peanut sIgE the greater the likelihood of clinical allergy.

[0263] In the PPOIT-003 randomised trial described in Example 3, serum levels of peanut sIgE were measured by ImmunoCap (Phadia) at baseline (TO), end-of-treatment (T1), 8 weeks post-treatment (T2) and 12 months post-treatment (T3). At baseline, the levels of sIgE were similar across the three treatment groups. The results are shown in Table 24, with groups compared in a pairwise fashion using the Wilcoxon Rank Sum.

[0264] At T1, peanut sIgE levels were significantly lower in the PPOIT group (6.6 kU/L) compared to the Placebo group (14.5 kU/L) (p=0.0460) and in the OIT group (3.2 kU/L) compared to the Placebo group (14.5 kU/L) (p=0.0057), but not significantly different between PPOIT and OIT groups (6.6 kU/L and 3.2 kU/L respectively, p=0.2484).

[0265] At T2, peanut sIgE levels declined further in both the PPOIT group (3.7 kU/L) and the OIT group (2.7 kU/L) and remained significantly lower compared with the Placebo group (18.0 kU/L) (p=0.009 and p=0.001, respectively). There was no significant difference in peanut sIgE levels between the PPOIT and OIT groups (p=0.3700).

[0266] The median changes from baseline in peanut sIgE at T1 for the PPOIT group (−5.0 kU/L) and the OIT group (−2.2 kU/L) were both significantly greater than for the Placebo group (0.3 kU/L) (p<0.0001 and p=0.0005, respectively). There was no significant difference in median change from baseline for peanut sIgE at T1 between PPOIT and OIT (p=0.2507). At T2, the median change from baseline in peanut sIgE remained significantly greater in the PPOIT group (−2.7 kU/L) and the OIT group compared to the Placebo group (2.4 kU/L) (both p<0.0001). There was no significant difference in median change from baseline in peanut sIgE between PPOIT and OIT groups (p=0.6331).

[0267] These results show that treatment with both PPOIT and OIT modulated the underlying peanut specific allergic immune response. Other studies of Peanut OIT have not shown significant reductions in peanut sIgE after 1-2 years of treatment, with reductions usually only being achieved after 4 or more years of treatment.

TABLE-US-00025 TABLE 24 Serum Peanut slgE levels before and after treatment in PPOIT-003 trial P value P value PPOIT OIT Placebo PPOIT vs OIT vs N = 79 N = 83 N = 39 Placebo Placebo sIgE T0 Baseline n 76 81 38 Median 11.1 11.0 18.0 0.942 0.438 Min, 0.1, 0.1, 0.2, Max 1115.8 1861.0 306.0 Q1, Q3 1.9, 92.5 2.0, 44.0 3.8, 59.7 sIgE T1 Timepoint n 62 63 32 Median 4.5 3.5 26.0 0.003 <0.0001 Min, 0.0, 00, 0.4, Max 903.3 867.6 532.6 Q1, Q3 1.3, 25.8 1.0, 18.8 9.1, 77.7 sIgE T2 Timepoint n 61 61 24 Median 4.3 2.9 14.8 0.038 0.007 Min, 0.1, 0.1, 0.0, Max 966.1 664.8 1259.0 Q1, Q3 1.1,23.0 0.7, 20.7 2.9, 112.8 sIgE T3 Timepoint n 65 61 23 Median 5.4 1.9 14.6 0.02 0.001 Min, 0.0, 0.1, 0.0, Max 714.0 419.0 596.0 Q1, Q3 0.8, 21.3 0.5, 15.5 2.1, 100.0 Change from Baseline, End-of- treatment n 61 62 31 Median −4.6 −2.2 4.1 <0.0001 <0.0001 Min, −407.7, −993.4, −58.5, Max 65.6 82.8 344.2 Q1, Q3 −64.9, −0.5 −15.3, 1.6 −0.5, 28.8 Change from Baseline, 8 weeks post- treatment n 59 60 23 Median −3.5 −2.1 2.2 <0.0001 <0.0001 Min, −408.7, −1196.2, −11.1, Max 380.7 27.6 1181.7 Q1, Q3 −38.5, −0.1 −16.1, −0.1 −0.6, 49.0 Change from Baseline, 12 months post- treatment n 62 60 23 Median −5.8 −4.1 −0.7 0.007 0.021 Min, −407.5, −1442.0, −156.9, Max 36.6 3.9 581.4 Q1, Q3 −42.1, −1.0 −20.9, −1.0 −10.4, 7.0

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