key: cord-0737560-ibb6yp2p
authors: De Boeck, I.; Cauwenberghs, E.; Spacova, I.; Gehrmann, T.; Eilers, T.; Delanghe, L.; Wittouck, S.; Bron, P. A.; Henkens, T.; Gamgami, I.; Simons, A.; Claes, I.; Marien, J.; Arien, K. K.; Bakokimi, D.; Loens, K.; Jacobs, K.; Ieven, M.; Bruijning-Verhagen, P.; Delputte, P.; Coenen, S.; Verhoeven, V.; Lebeer, S.
title: Evaluation of a throat spray with lactobacilli in COVID-19 outpatients in a randomized, double-blind, placebo-controlled trial for symptom and viral load reduction
date: 2022-03-20
journal: nan
DOI: 10.1101/2022.03.17.22272401
sha: 4caeb89cc0fcdd83725189b2e5d2eed294b0ac35
doc_id: 737560
cord_uid: ibb6yp2p

Objectives: Primary care urgently needs treatments for COVID-19 patients because current options are limited, while these patients account for more than 90% of the people infected with SARS-CoV-2. Methods: We evaluated a throat spray containing three Lactobacillaceae strains with broad antiviral properties in a randomized double-blind placebo-controlled trial. Seventy-eight eligible COVID-19 patients were randomized to verum (n=41) and placebo (n=37) within 96 hours of positive PCR-based SARS-CoV-2 diagnosis and per-protocol analysis was performed. Symptoms and severity were reported daily via an online diary. Combined nose-throat swabs and dried blood spots were collected at regular time points in the study. Results: The daily reported symptoms were highly variable, with no added benefit for symptom resolution in the verum group. Specific monitoring of the applied lactobacilli strains showed that they were detectable via microbiome (27%) and qPCR analysis (82%) of the verum group. Their relative abundances were also negatively correlated with the acute symptom score. At the end of the trial, a trend towards lower SARS-CoV-2 viral loads was observed for the verum group (2/30, 6.7% positive) compared to the placebo group (7/27, 26% positive) (p = 0.07). Conclusions: Despite a trend towards lower SARS-CoV-2 viral loads at the end of the trial and a negative correlation between relative abundances of the applied lactobacilli in the microbiome and acute symptoms, we did not observe a significant effect on overall symptom score for the verum group. This suggests that studies with earlier application of the spray in larger study populations are needed to further assess application potential.

SARS-CoV-2 diagnosis and per-protocol analysis was performed. Symptoms and severity were reported 23 daily via an online diary. Combined nose-throat swabs and dried blood spots were collected at regular 24 time points in the study. 25 . CC-BY-NC-ND 4.0 International license It is made available under a perpetuity.

is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint these milder cases exert a significant burden on healthcare professionals in primary care [2, 3] . In 44 addition, asymptomatic and presymptomatic transmission is the main driver for to others [4] . 45

Respiratory viral infections can have severe health consequences due to imbalanced immune 46 activation and bacterial co-infections associated with airway tissue disruption and severe inflammation 47 [5] . This clearly shows the urge for more treatment and/or prevention options in COVID-19 48 outpatients, which can improve different aspects of the disease: symptom relieve, transmission 49 reduction and decreased hospitalization. 50 . CC-BY-NC-ND 4.0 International license It is made available under a perpetuity.

is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint

The copyright holder for this this version posted March 20, 2022. ; https://doi.org/10.1101/2022.03.17.22272401 doi: medRxiv preprint 3 Microbiome or probiotic therapy is an emerging alternative treatment option for respiratory viral 51 diseases based on the potential multifactorial action of beneficial bacteria in the airways [6] . While 52 oral administration of such microbiome therapeutics or probiotics remains most common [7] , this 53 route relies on systemic effects to ameliorate respiratory infections. Also during the current COVID-19 54 pandemic, oral administrations targeting the gut have already been explored [8] [9] [10] . Alternatively, 55 topical application of rationally-selected probiotics in the airways might offer several advantages [11] , 56 as this could lead to direct blocking or inhibition of respiratory viruses [12] , and direct immune 57 modulation at the site of infection and inflammation [13, 14] . Indeed, the probiotic definition is not 58 limited to the gut [15] . We recently developed a microbiome-modulating throat spray with three 59

Lactobacillaceae strains that were selected based on their safety and in vitro multifactorial modes of 60 action on the key aspects of viral infection and disease, and their ability to thrive in the human 61 respiratory tract of healthy volunteers [16] . Yet, microbiome therapy with live bacteria has several 62 challenges, such as formulation and selection of target patient population. 63

Here, we evaluated the clinical potential of this throat spray with Lacticaseibacillus casei AMBR2, 64

Lacticaseibacillus rhamnosus GG, and Lactiplantibacillus plantarum WCFS1 against COVID-19 in a 65 randomized, double-blind, placebo-controlled trial in COVID-19 outpatients exhibiting mild-to-66 moderate symptoms. Specifically, we monitored impact on symptom severity, time to improvement, 67 viral load, anti-SARS-CoV-2 antibodies and the respiratory microbiome in an out-of-hospital setting. 68

This trial relied on self-sampling and included collection of combined nose-throat swabs, fingerprick 69 blood samples and reporting of symptom and severity via an online diary. 70

Clinical trial design: A double-blind, placebo-controlled clinical trial was performed with a microbiome 72 throat spray in COVID-19 outpatients within 96 hours after a positive PCR test in government facilities 73 (see more details in supplementary methods). Approval was obtained from the committee of medical 74 Ethics (UZA/UAntwerpen, B3002021000018 and NCT04793997). Informed consent was obtained from 75 . CC-BY-NC-ND 4.0 International license It is made available under a perpetuity.

is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint Study procedures: Patients were asked to use the verum spray or placebo for 14 days, with one week 79 of follow-up, and filled in an online diary via Qualtrics (Qualtrics, Provo, UT, USA) (detailed description 80 in Figure S1 ). Ten common COVID-19 symptoms were monitored, according to [17] . is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint

The copyright holder for this this version posted March 20, 2022. ; https://doi.org/10.1101/2022.03.17.22272401 doi: medRxiv preprint 5 meier survival analysis was performed using the R survival package. The symptomatic tipping point 101 was taken as the event, and the time until occurrence was tested in the different treatment groups. 102

Differences between symptom scores in covid-positive and -negative participants (based on PCR) were 103 tested using a random effect model, symptom ~ covid + (1|participant). P-values were adjusted for 104 multiple testing using Bonferroni. Differential bacterial abundances between treatment groups were 105 tested with a random effect model with CLR(ra) ~ treatment + plate + qubit_score + library_size + 106 (1|participant), where CLR(ra) is the centered-log-ratio transformed relative abundance of a given 107 bacteria, and plate, qubit_score and library size constitute technical confounders. Effect sizes for the 108 treatment group were calculated for timepoints T2 and T3, at which the participants were using the 109 spray. 110

The trial was conducted from February 24, 2021 to April 30, 2021 at the University of Antwerp. Table 1 . 120

The sprays were overall well-tolerated, but participants in both study groups often reported 121 unpleasant taste (mainly verum group) or texture (verum and placebo) of the spray. Also for the online 122 diaries, the compliance was high: the median number of completed diaries was 20/21 days, with 31.3% 123 of the study population showing full compliance, with filling in the diary every days. The compliance of 124 . CC-BY-NC-ND 4.0 International license It is made available under a perpetuity.

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The copyright holder for this this version posted March 20, 2022. ; https://doi.org/10.1101/2022.03.17.22272401 doi: medRxiv preprint 6 the self-sampling was 80.5% (509/632) for the combined nose/throat swabs, and 83.5% (132/158) for 125 the fingerprick blood samples. 126

Symptoms at the start of the study are shown in Table 1 . Cough (68%), runny/blocked nose (70%), 129 headache (65%) and fatigue (75%) were most frequently reported. The average total symptom score 130 at start of the study was 13.4 ± 8.6 in the verum group and 15.2 ± 9.3 in the placebo group (difference 131 not significant) (Table S1 ). 132

Severity of the symptoms was evaluated between both treatment groups over the study via the 133 distribution of the different severity scores (total, URT, acute and symptom) at every day (see also 134 Table S1 ). The same tendency for the verum and placebo group was observed with no significant 135 differences ( is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint 

Principal coordinates analysis (PCoA) showed no major shifts in the overall nose/throat microbial 161 composition for the time points nor for the microbiome treatment ( Figure 4A ). However, specific 162 effects on abundances of certain taxa were observed. When focusing on abundances of the amplicon 163 sequence variants (ASVs) of the Lactobacillaceae strains administered with the throat spray, significant 164 differences were observed between verum and placebo groups at different time points with mean 165 relative abundances for the L. casei ASV, L. plantarum ASV and L. rhamnosus ASV in the verum group 166 found to be 1.6%, 1.3% and 0.5%, respectively, over the entire study ( Figure 4B , Table S3 ). In the 167 placebo group, these numbers were below 0.01% for all three ASVs (see also Table S3 ). Prevalences 168 (presence) based on MiSeq data were 38.6%, 28% and 13.4% for the L. casei ASV, L. plantarum ASV 169 and L. rhamnosus ASV, respectively, while this was 10.5%, 7% and 2% in the placebo group, pointing 170 at the fact that the related taxa to the applied lactobacilli are also endogenously present, but in low 171 numbers. Therefore, the presence of the specifically applied Lactobacillaceae strains was also 172 confirmed via qPCR with clear difference between verum (on average detected in 82% of the study 173 population) and placebo (on average 21%) and also between the estimated CFU/ml counts of the three 174 is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint

The copyright holder for this this version posted March 20, 2022. ; https://doi.org/10.1101/2022.03.17.22272401 doi: medRxiv preprint shortness of breath on day 8 (p = 0.02) and day 12 (p = 0.04) compared to placebo [23] . However, it 225 should be noted that the authors did not report any correction for multiple testing in this study, which 226 is also not yet peer reviewed. In another larger trial (n = 300) with an oral probiotic mixture of L. 227 plantarum strains and Pediococcus acidilactici KABP021, patients in the verum group reported fewer 228 days of fever, cough, headache, body aches and shortness of breath [8] . is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint

The copyright holder for this this version posted March 20, 2022. ; https://doi.org/10.1101/2022.03.17.22272401 doi: medRxiv preprint Belgium; ID: BE 71030031000). 303

IDB, IS, IC, TH and SL are inventors on a patent application (BE2021/5643) related to this work. IC, TH, 305 IG and AS are working at YUN NV (www.yun.be) who formulated the spray for this study. SL is a 306 member of the scientific advisory board of YUN NV. The PhD research of LD is currently funded by 307 VLAIO through a Baekeland mandate in collaboration with YUN NV. YUN was not involved in the clinical 308 study design or data analysis of this work. PAB is a consultant for multiple companies in the food and 309 health industry, but they were not involved in this manuscript. The remaining authors have no conflicts 310 of interest to declare related to this work. 311

All data produced in the present study are available upon reasonable request to the authors or will be 313 deposited online after publication 314 is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint

The copyright holder for this this version posted March 20, 2022. ; https://doi.org/10.1101/2022.03.17.22272401 doi: medRxiv preprint

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Probiotic improves symptomatic and viral clearance in Covid19 339 outpatients: a randomized, quadruple-blinded, placebo-controlled trial

Bacteriotherapy in Patients With COVID-19: A Retrospective Cohort Study. Front Nutr

Challenges in the Management of SARS-CoV2 Infection: The Role of Oral Bacteriotherapy as 348

Complementary Therapeutic Strategy to Avoid the Progression of COVID-19

is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprintThe copyright holder for this this version posted March 20, 2022. ; https://doi.org/10.1101/2022.03.17.22272401 doi: medRxiv preprint