key: cord-0890448-wyebf5iv authors: Yamamoto, K.; Hosogaya, N.; Inoue, T.; Jounai, K.; Tsuji, R.; Fujiwara, D.; Yanagihara, K.; Izumikawa, K.; Mukae, H. title: Efficacy of Lactococcus lactis strain plasma (LC-Plasma) in easing symptoms in patients with mild coronavirus disease 2019 (COVID-19): protocol for an exploratory, multicenter, double-blinded, randomized controlled trial (PLATEAU study) date: 2022-02-15 journal: nan DOI: 10.1101/2022.02.13.22270913 sha: 7ae3c750ca54beda88e48ac298df28b9d8cc0f71 doc_id: 890448 cord_uid: wyebf5iv Introduction: The coronavirus disease 2019 (COVID-19) pandemic has been a major concern worldwide; however, easily accessible treatment options for patients with mild COVID-19 remains limited. Since oral intake of Lactococcus lactis strain Plasma (LC-Plasma) enhances both the innate and acquired immune systems through activation of plasmacytoid dendritic cells (pDCs), we hypothesized that the oral intake of LC-Plasma could aid the relief or prevention of symptoms in patients with asymptomatic or mild COVID-19. Methods and analysis: This is an exploratory, multicenter, double-blind, randomized, placebo-controlled trial. This study was initiated in December 2021 and concludes in April 2023. The planned number of enrolled subjects is 100 (50 patients x 2 groups); subject enrolment will be conducted until October 2022. Patients with asymptomatic or mild COVID-19 will be enrolled and randomly assigned in a 1:1 ratio to Group A (oral intake of LC-Plasma-containing capsule, 200 mg/day, for 14 days) or Group B (oral intake of placebo capsule, for 14 days). The primary endpoint is the change in subjective symptoms measured by the severity score. Secondary endpoints include SARS-CoV-2 viral loads, biomarkers for pDC activation, serum SARS-CoV-2-specific antibodies, serum cytokines, interferon and interferon-inducible antiviral effectors, and the proportion of subjects with emergency room visits to medical institutions or who are hospitalized. Ethics and dissemination: The study protocol was approved by the Clinical Research Review Board of Nagasaki University, in accordance with the Clinical Trials Act of Japan. The study will be conducted in accordance with the Declaration of Helsinki, Clinical Trials Act, and other current legal regulations in Japan. Written informed consent will be obtained from all participants. The results of this study will be reported in journal publications. Registration: This study was registered in the Japan Registry of Clinical Trials (registration number: jRCTs071210097). 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 February 15, 2022. ; https://doi.org/10.1101/2022.02.13.22270913 doi: medRxiv preprint this study will be reported in journal publications. 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 February 15, 2022. ; https://doi.org/10.1101/2022.02.13.22270913 doi: medRxiv preprint Strengths and limitations of this study 52  This is the first randomized controlled trial to assess the efficacy of Lactococcus lactis strain 53 Plasma (LC-Plasma) in preventing the onset and attenuation of symptoms in patients with 54 asymptomatic or mild COVID-19. 55 This study is also the first to evaluate the significance of pDC-related immune responses, 56 including interferon production, clearance of symptoms, and prevention of COVID-19 57 progression. 58 The results of this study may contribute to the development of novel treatment options for 59 asymptomatic or mild COVID-19 patients. 60  This is an exploratory study, due to the lack of previous clinical evidence that evaluated the effect 61 of LC-Plasma intake in patients with COVID-19. 62 Other limitations include the subjective endpoint as the primary endpoint and generalizability, 63 since this study will be conducted only in Japan in Japanese patients. 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 February 15, 2022. ; https://doi.org/10.1101/2022.02.13.22270913 doi: medRxiv preprint The coronavirus disease 2019 (COVID-19) pandemic is a major concern worldwide. In Japan, a 67 cumulative total of 2.92 million polymerase chain reaction-positive cases have been confirmed, and 68 18,387 deaths have been reported by the Ministry of Health, Labor and Welfare in Japan as of 69 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 February 15, 2022. ; https://doi.org/10.1101/2022.02.13.22270913 doi: medRxiv preprint 8 Thus, LC-Plasma is a safe and easy-to-use candidate for treating asymptomatic or mild COVID-19. 102 The present study aimed to evaluate the efficacy of LC-Plasma capsules in preventing the onset and 103 attenuating symptoms of COVID-19 in patients with asymptomatic or mild infection. 104 The "efficacy of Lactococcus lactis strain PLasmA To EAse symptoms in patients with coronavirUs 108 disease 2019 (PLATEAU) study: a multicenter, double-blinded, randomized placebo-controlled trial" Nagasaki Hospital, Kouseikai Hospital, and Nagasakikita Tokushukai Hospital. As shown in Figure 1 , 117 eligible patients will be asked to participate in this study, and informed consent will be obtained prior 118 to registration/randomization. After written consent is obtained from the eligible patients, they will be 119 . CC-BY-NC 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 February 15, 2022. ; https://doi.org/10.1101/2022.02.13.22270913 doi: medRxiv preprint 9 enrolled and randomized into Group A (intake of LC-Plasma-containing capsule) or Group B (intake 120 of placebo capsule). Group A patients will be treated with 200 mg/day of heat-killed LC-Plasma, which 121 includes at least 4.0 × 10 11 cells. 122 123 A LC-Plasma-containing capsule contained 50 mg heat-killed LC-Plasma, 127.3 mg dextrin, and 2.7 125 mg calcium stearate. A placebo capsule contained 177.3 mg dextrin and 2.7 mg calcium stearate. 126 Subjects will orally ingest 4 test capsules (LC-Plasma-containing capsules or placebo capsules) once 127 daily. The study controller has confirmed that the two types of test capsules cannot be distinguished 128 on the basis of taste, appearance, or smell. 129 130 Patients who are SARS-CoV-2 positive with asymptomatic or mild COVID-19 disease and who are 132 stayed at isolation facilities for COVID-19 patients in Nagasaki City, Nagasaki, Japan are eligible for 133 this study. The detailed inclusion criteria are as follows: 1) subjects who are SARS-CoV-2 positive, 2) 134 whose arterial oxygen saturation (SpO2) is 96% or higher, 3) who are aged 20 years or older and 135 younger than 65 years, 4) who do not refuse to disclose their vaccination status, 5) who can be stayed 136 at isolation facilities for COVID-19 patients designated by Nagasaki City, and 6) who give their written 137 . CC-BY-NC 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 February 15, 2022. ; https://doi.org/10.1101/2022.02.13.22270913 doi: medRxiv preprint consent to participate in the study. Subjects who meet any of the following exclusion criteria will be 138 excluded from participation: 1) obese (body mass index (BMI) ≥30 kg/m 2 ), 2) subjects with strong 139 dyspnea, chest pain, or hemosputum, 3) previous history of COVID-19, 4) being treated or planning 140 to be treated with neutralizing antibody drugs for SARS-CoV-2, 5) being treated with 141 immunosuppressive agents, antirheumatic agents, corticosteroids, or immunoglobulin preparations, 6) 142 subjects administered oral intestinal regulators, 7) taking one or more beverage or food containing LC-143 Plasma or yogurt that contains Lactobacillus delbrueckii subsp. bulgaricus daily, 8) who are pregnant, 144 possibly pregnant, or breastfeeding, 9) participating in other clinical trials, 10) requiring legal 145 representation for giving consent, or 11) with other conditions that the responsible investigator or sub-146 investigators deem inappropriate for study participation. 147 148 The informed consent document (see supplementary material) will be provided to candidates who 150 meet all inclusion criteria and none of the exclusion criteria to provide a comprehensive explanation 151 of this study. Written consent will be obtained from all participants. After obtaining consent, 152 candidates will be provisionally enrolled in this study. Additional candidates will be enrolled in this 153 study when they enter isolation facilities as described above. 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 February 15, 2022. ; https://doi.org/10.1101/2022.02.13.22270913 doi: medRxiv preprint After obtaining informed consent, eligible participants will be randomly assigned in a 1:1 ratio to 157 Group A (intake of LC-Plasma-containing capsule, 200 mg/day) or Group B (intake of placebo 158 capsule). The randomization sequence will be generated using a computer-based dynamic allocation 159 method with a minimization procedure to balance allocation factors (age: less than 50 years or 50 160 years or older; SARS-CoV-2 vaccination status; use of anti-SARS-CoV-2 agents). 161 162 This study will be conducted as a double-blinded trial. All parties are blinded, including 164 investigators, participants, the manufacturer of test capsules, core laboratories, and biostatisticians. A 165 central registration number will be used to identify subjects for anonymization. The manufacturer of 166 test capsules marked LC-Plasma-containing capsules and placebo capsules with specific characters 167 such as X and Y, and the person in charge of assignment will complete a corresponding table of 168 characters and central registration numbers. The computer-based allocation system will provide a 169 central registration number linked to each subject during registration/assignment. Unblinding will be 170 conducted only in emergency cases such as the occurrence of serious adverse events (SAEs). 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 February 15, 2022. ; https://doi.org/10.1101/2022.02.13.22270913 doi: medRxiv preprint Subjects will be assigned to a group and orally administered test capsules for 14 days. All subjects 174 will be subsequently observed for 14 days (observation points are days 1, 4, 8, and 14) . During the 175 observation period, subjects will be restricted to using systemic antiviral agents (except topical 176 antiviral agents) or intestinal regulators. For palliative care for COVID-19, temporary use of 177 antitussive agents, expectorants, general cold medications, antipyretic analgesic (acetaminophen only), 178 and antidiarrheal agents are allowed, but not for routine use. In addition, during the observation period, 179 addition, discontinuation, switch, or dose change of any medical agents or supplements will be 180 recorded. Furthermore, subjects will be asked to refrain from consuming lactic acid bacteria-181 containing foods, such as yogurt, during the observation period. The intervention will be discontinued 182 if the subjects meet any of the discontinuation criteria: consent withdrawal, worsening of the primary 183 disease, complications, adverse events (AEs) requiring discontinuation of the study intervention, 184 remarkably poor adherence to the test capsules regimen, or any other condition that investigators deem 185 appropriate for discontinuation of the study intervention. 186 Table 1 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 primary endpoint of this study is the change in subjective symptoms measured using the 215 severity score [21] and Visual Analogue Scale (VAS). Subjective symptoms (cough, shortness of 216 breath, fatigue, headaches, anosmia, dysgeusia, and anorexia) are assessed using a 4-point Likert scale 217 (not affected = 0 points, little effect = 1 point, affected = 2 points, and severely affected = 3 points), 218 and the total severity score is calculated as the sum of scores for these seven subjective symptoms. 219 The severity score questionnaire is shown in Supplementary Table 1 . A case report form will be used for data collection. Data collection and management will be carried 228 out by third party entities to avoid bias. Data will be managed by Soiken Inc., the Data Management 229 Group (Data Center). To ensure quality, the study will be monitored by Soiken Inc., the Monitoring 230 . CC-BY-NC 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 February 15, 2022. ; https://doi.org/10.1101/2022.02.13.22270913 doi: medRxiv preprint 232 During this study, investigators will constantly monitor for any AEs during regular medical 234 checkups. All related AEs, including study agent side effects, abnormal clinical laboratory test values, 235 and untoward medical occurrences, will be reported and documented. If AEs meet the following 236 criteria, they are referred to as SAEs based on the ICH E2A, ICH E2D, and "Ethical Guidelines for 237 Medical and Health Research Involving Human Subjects": [22] AEs that result in 1) death, 2) life-238 threatening hospitalization, or 3) extension of hospitalization, 4) persistent or significant disability or 239 incapability, 5) medically important or critical condition, 6) AEs that are equivalently severe to criteria 240 1) to 5), or 7) AEs that cause congenital abnormality or birth defects. AEs will be followed until 241 normalization or recovery to a level that is not considered an AE. 242 243 The severity score was used in a case series study to estimate the patients' subjective symptoms. When the total severity score from this previous study was translated using a calculation method 246 mentioned in the outcomes section of the present study, the total score on days 0 and 14 was 11 ± 5.3 247 and 1.7 ± 2.3, respectively, and the change from day 0 to day 14 was -9.3 ± 6.0. 248 Since this study will enroll patients who are asymptomatic or have mild COVID-19, we assumed 249 . CC-BY-NC 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 February 15, 2022. ; https://doi.org/10.1101/2022.02.13.22270913 doi: medRxiv preprint that the severity score of cough at baseline (day 1) will be 0 to 1 and that of other symptoms will be 0 250 in this study. Therefore, the mean total severity score at baseline predicted in this study is 0.5. We also 251 assumed that the severity score will not worsen in Group A (i.e., change in the total severity score = 252 0). Since a recent meta-analysis reported that 48.9% of initially asymptomatic patients with COVID-253 19 became symptomatic,[23] we assumed that the severity score in Group B will worsen to a level of 254 half of the severity score at day 0 reported in the case series study mentioned above,[21] meaning that 255 the total severity score would be 5.5 (i.e., change in the total severity score = 5). Since it is expected 256 that the degree of subjective symptoms varies widely among patients when asymptomatic subjects 257 become symptomatic, we assumed that the standard deviation in the change in the severity score will 258 be 7.0, which is slightly larger than that in the case series study mentioned above. [21] Under these 259 assumptions, the minimum sample size required to achieve a significance of 0.05 from a two-sided 260 test with a statistical power of 90% was determined to be 42 subjects for both groups, or a total of 84 261 subjects. We estimated the dropout rate to be 15%; thus, planned enrolment was set at 100 subjects, 262 with 50 in each group. 263 264 All tests will be two-sided, and a p-value <0.05 will be considered statistically significant. As this 266 is an exploratory trial, multiplicity will not be adjusted for all endpoints. A statistical analysis plan was 267 . CC-BY-NC 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 February 15, 2022. ; https://doi.org/10.1101/2022.02.13.22270913 doi: medRxiv preprint developed prior to the database lock. All statistical analyses will be conducted by independent 268 biostatisticians. 269 Three analysis sets are defined in this study; the full analysis set (FAS) includes all patients who 270 will be registered in this study. However, patients with severe protocol violations, such as registration 271 without obtaining consent or registration outside of the enrolment period, will be excluded from the 272 FAS. The per-protocol set excludes patients with a protocol violation, such as violation of eligibility 273 criteria, use of prohibited or restricted concomitant treatments, or poor adherence to the test capsules 274 (less than 75% or more than 120%). The safety analysis set includes all patients who will be registered 275 in this study who receive at least one dose of the test capsules. 276 Patient characteristics at baseline will be presented as frequencies and proportions for categorical 277 data and summary statistics (number of patients, mean, standard deviation, minimum, first quartile, 278 median, third quartile, and maximum) for continuous data. Patient characteristics will be compared 279 using the chi-square test or Fisher's exact test for categorical data and the two-sample t-test or 280 Wilcoxon rank sum test for continuous variables. 281 The primary endpoint of this study is the change in subjective symptoms measured by the severity 282 score. [21] The total score of the severity score is calculated by summarizing the severity scores of 283 seven symptoms, and the summary statistics of change in the total score of the severity score from 284 baseline to day 14 will be calculated. Analysis of covariance (ANCOVA) will be conducted to test the 285 . CC-BY-NC 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 February 15, 2022. ; https://doi.org/10.1101/2022.02.13.22270913 doi: medRxiv preprint null hypothesis that the change in the total score of the severity score from baseline to day 14 is the 286 same in both groups. The allocation factors age (less than 50 years or 50 years or older), vaccination 287 status for SARS-CoV-2 (vaccinated or unvaccinated), use of anti-SARS-CoV-2 agent (presence or 288 absence of anti-SARS-CoV-2 agent use), and the total score of the severity score at baseline will be 289 used as covariates in the ANCOVA. The sensibility analysis will be performed using the mixed-effects 290 model for repeated measures with an unstructured covariance structure with treatment groups, time 291 (day), interactions between treatment groups and time, allocation factors, and the total score of the 292 severity score at baseline as fixed effects, and subjects as random effects. If the calculation results do 293 not converge, compound symmetry will be used. The VAS score will be analyzed similar to the 294 analysis of severity score. 295 For the secondary endpoints, summary statistics (number of patients, mean, standard deviation, 296 minimum, first quartile, median, third quartile, and maximum) for measurements, changes from 297 baseline, and percentage changes from baseline will be calculated for continuous data. Frequencies 298 and proportions will be calculated for categorical data. Two-sample t-test or Wilcoxon rank sum test 299 for intergroup comparisons of continuous data, one-sample t-test or Wilcoxon signed-rank test for 300 intragroup comparisons of continuous data, and the chi-square test or Fisher's exact test for intergroup 301 comparisons of categorical data will be performed. 302 For safety endpoints, summary statistics for the frequency of AEs will be calculated for each group, 303 . CC-BY-NC 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 February 15, 2022. ; https://doi.org/10.1101/2022.02.13.22270913 doi: medRxiv preprint 21 and Fisher's exact tests will be performed for intergroup comparisons. 304 305 Patients and the public were not involved in the conception or planning of this study and will not 307 be involved in the execution, analysis, and evaluation. 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 February 15, 2022. Safety monitoring Ministry of Health, Labour and Welfare in Japan. Novel Coronavirus (COVID-19) World Health Organization. WHO Coronavirus (COVID-19) Dashboard. Available at https World Health Organization. Classification of Omicron (B.1.1.529): SARS-CoV-2 Variant of 394 529)-sars-cov-2-variant-of-concern Ministry of Health, Labour and Welfare in Japan. 11 things you need to know NOW about 397 COVID-19 REGN-COV2, a Neutralizing Antibody 401 Outpatients with Covid-19 Effectiveness of Severe Acute Respiratory Syndrome 403 Coronavirus 2 Monoclonal Antibody Infusions in High-Risk Outpatients Use of monoclonal antibody therapy for nosocomial 406 SARS-CoV-2 infection in patients at high risk for severe COVID-19: experience from a 407 tertiary-care hospital in Germany Molnupiravir-A Novel Oral Anti-SARS-CoV-2 Agent antiviral for COVID-19, Lagevrio (molnupiravir), approved by MHRA. Available at https Spherical lactic acid bacteria activate plasmacytoid 415 dendritic cells immunomodulatory function via TLR9-dependent crosstalk with myeloid 416 dendritic cells lactic acid bacterium that activates plasmacytoid dendritic cells Natural interferon alpha/beta-producing cells link 420 innate and adaptive immunity Effects of oral intake of plasmacytoid dendritic 422 cells-stimulative lactic acid bacterial strain on pathogenesis of influenza-like illness and 423 immunological response to influenza virus Administration of plasmacytoid dendritic cell-stimulative 425 lactic acid bacteria enhances antigen-specific immune responses Overview of Immune Response During SARS-CoV-2 428 Infection: Lessons From the Past Type I and Type III Interferons -Induction, Signaling, Evasion, and 430 Application to Combat COVID-19 Antigen-Specific Adaptive Immunity 432 to SARS-CoV-2 in Acute COVID-19 and Associations with Age and Disease Severity The safety evaluation of long-term or excessive 435 LC-Plasma reported no safety concerns. In addition, LC-Plasma-containing yogurt, beverages, and 322 supplements have been commercially available since 2012, and no health hazards associated with LC-323Plasma-containing products have been reported. This suggests the safety of the LC-Plasma-containing 324 supplements used in this study (200 mg daily for 14 days). Molnupiravir, the first approved oral 325 antiviral agent for high-risk COVID-19 patients, has common side effects including diarrhea, nausea, 326 dizziness, and headache within 14 days of the last dose, and is restricted in pregnant women because 327 it affects the development of the fetus [24, 25] . Non-elderly and non-high-risk patients with mild 328 COVID-19 rarely progress to severe disease; therefore, it is reasonable to choose safer drugs that 329 sufficiently control their symptoms. 330This study has several limitations. First, this is an exploratory study due to the lack of previous 331 clinical evaluation of the effects of LC-Plasma intake in patients with COVID-19. The target number 332 of enrolled patients in this study was calculated from results of a case series study estimating subjective 333 symptoms in non-hospitalized patients with COVID-19 treated with the histamine-2 receptor 334 antagonist famotidine.[21] Since this case series evaluated subjective symptoms in only 10 patients, 335 the calculated mean and standard deviation of the results might not reflect the actual patients' 336 subjective symptoms. Second, the primary endpoint in this study is patients' subjective symptoms as 337 reported by subjects themselves. Therefore, biases such as responder bias, non-responder bias, and 338 volunteer bias cannot be completely avoided. However, the effect of bias is minimized using a double-339 . CC-BY-NC 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) preprintThe copyright holder for this this version posted February 15, 2022. ; https://doi.org/10.1101/2022.02.13.22270913 doi: medRxiv preprint 23 blinded study design. Third, this study will be conducted in medical institutions in Japan and will 340 enroll only Japanese patients. These constraints could limit the generalizability of this study. Further University prior to the implementation of this study according to the amended protocol. Written 350 informed consent will be obtained from all participants after a full explanation of the study. Any health 351 hazards caused by this study will be compensated by clinical research insurance. The results of this 352 study will be disseminated through medical conferences and journal publications. 353Datasets generated and/or analyzed during this study will not be publicly available because of the 354 absence of a statement in the study protocol and the informed consent documents enabling data sharing 355 with a third party after the end of the study. Data sharing has not been approved by the certified review 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 February 15, 2022. 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 February 15, 2022. 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 February 15, 2022. ; https://doi.org/10.1101/2022.02.13.22270913 doi: medRxiv preprint 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 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 February 15, 2022. ; https://doi.org/10.1101/2022.02.13.22270913 doi: medRxiv preprint