Efficacy of Lactococcus lactis Strain Plasma in Patients with Mild COVID-19: A Multicenter, Double-Blinded, Randomized-Controlled Trial (PLATEAU Study)

Abstract

Introduction

Coronavirus disease 2019 (COVID-19), caused by Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), is still an ongoing public health threat. COVID-19 can be accompanied by prolonged symptoms, known as “long COVID”, however, no pharmaceutical treatments are currently available for these symptoms. Lactococcus lactis strain Plasma (LC-Plasma; Lactococcus lactis subsp. lactis JCM 5805) directly activates human plasmacytoid dendritic cells (pDCs) and triggers antiviral immune responses. We hypothesized that LC-Plasma reduced SARS-CoV-2 viral load and eased symptoms in patients with mild COVID-19.

Methods

This PLATEAU study enrolled 100 patients with mild COVID-19 during Omicron BA.1 endemic, who were randomized into the LC-Plasma or placebo group in a 1:1 ratio and were observed for 14 days. The primary endpoint was change in total score of eight subjective symptoms (fatigue, anorexia, headache, cough, shortness of breath, chest pain, smell, and taste disturbance). Secondary endpoints included each symptom, SARS-CoV-2 viral load, and pDCs.

Results

The primary endpoint did not show between-group differences. However, the proportion of patients without smell and taste disturbances was significantly higher in the LC-Plasma group on day 13 (p = 0.030). The LC-Plasma group showed a significantly earlier decrease in SARS-CoV-2 viral load on day 4 (p < 0.001) and an increase in pDCs on day 8 (p = 0.0498). Mild adverse events, such as diarrhea, cough-variant asthma, and urticaria, occurred in three (5.9%) patients in the LC-Plasma group.

Conclusions

The intake of LC-Plasma in patients with mild COVID-19 activates pDC, decreases SARS-CoV-2 viral load earlier, and may improve smell and taste disorders more quickly. LC-Plasma could be a safe, inexpensive, and easily accessible tool for the treatment of mild COVID-19.

Trial Registration

jRCTs071210097.

Graphical Abstract

Supplementary Information

The online version contains supplementary material available at 10.1007/s40121-025-01246-8.

Key Summary Points

Why carry out this study?

COVID-19 can be accompanied by prolonged symptoms—so-called “long COVID”; however, no pharmaceutical treatments are currently available for these symptoms.

Lactococcus lactis strain Plasma (LC-Plasma; Lactococcus lactis subsp. lactis JCM 5805) has been reported to directly activate human plasmacytoid dendritic cells (pDCs) and trigger antiviral immune responses.

We hypothesized that LC-Plasma reduces SARS-CoV-2 viral load and eases symptoms in patients with mild COVID-19.

What was learned from the study?

The intake of LC-Plasma in patients with mild COVID-19 activated pDC, decreased SARS-CoV-2 viral load earlier, and improved smell and taste disorders more quickly.

LC-Plasma can be a safe, inexpensive, and easily accessible tool to activate innate immune response and ease symptoms in patients with mild COVID-19.

Digital Features

This article is published with digital features, including a graphical abstract, to facilitate understanding of the article. To view digital features for this article, go to 10.6084/m9.figshare.30146173.

Introduction

Coronavirus disease 2019 (COVID-19) is caused by Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2). It was associated with approximately 244,000 deaths in the U.S. [1] and 48,000 in Japan between January and December 2022 [2]. Current concerns during the Omicron wave include high transmissibility, unresolved long COVID, and rising mortality across countries [3], underscoring its ongoing public health threat. Although several antiviral agents are used for the Omicron strains, these are mainly administered patients with moderate-to-severe or high-risk mild COVID-19. Smell and taste dysfunction are hallmark long COVID symptoms [4], with one-third persisting beyond a year [5]; however, no pharmaceutical treatments are currently available. There remains a need for accessible and affordable treatment options for patients with mild COVID-19 during the Omicron wave.

Lactococcus lactis strain Plasma (LC-Plasma; Lactococcus lactis subsp. lactis JCM 5805) is a lactic acid bacterium that directly activates human plasmacytoid dendritic cells (pDCs), inducing type I and III interferons (IFNs) [6, 7] and triggering antiviral immune responses. Unlike other lactic acid bacteria, LC-Plasma uniquely activates pDCs [8]. It has shown protective effects against viral infections. In vivo, LC-Plasma reduced the severity of parainfluenza [9] and dengue virus infection [10]. Human trials have shown that LC-Plasma intake activates pDCs [11], increases IFN-α gene expression [12], and alleviates common cold symptoms [12, 13]. Moreover, it reduced influenza incidence in Japan [14] and dengue-like symptoms in Malaysia [15]. LC-Plasma boosts secretory immunoglobulin A in saliva and maintains pDC activation [13], leading to two antiviral responses: (1) IFN-α/β production to inhibit viral replication [16], and (2) T- and B-cell activation for adaptive immunity [17]. During SARS-CoV-2 infection, early-phase IFN production is a key immune response [18]. Recently, in vitro studies showed that LC-Plasma-stimulated pDCs suppressed SARS-CoV-2 replication [19]. Based on these, we hypothesized that LC-Plasma intake may reduce SARS-CoV-2 load and symptoms in patients with COVID-19. This study represents the first clinical trial of LC-Plasma in viral infection.

Methods

Study Design and Ethical Approval

This “Efficacy of Lactococcus lactis strain PLasmA (LC-Plasma) To EAse symptoms in patients with coronavirUs disease 2019 (COVID-19); multi-center, double-blinded, randomized placebo-controlled trial”, designated as a PLATEAU study, was conducted at seven medical institutions in Nagasaki, Japan. The study protocols were inspected and approved (approval no. CRB21-009) in November 2021 by the Clinical Research Review Board of Nagasaki University. The study was registered with the Japan Registry of Clinical Trials (jRCT) (registration number: jRCTs071210097) in December 2021. The trial protocol has been described previously [20]. Patient enrollment was conducted from December 2021 to April 2022. The study was conducted in accordance with the Declaration of Helsinki, Clinical Trials Act, and other current legal regulations in Japan. Written informed consent was obtained from all enrolled patients who met the eligibility criteria before the intervention. To avoid bias and ensure quality, a third-party entity (Soiken, Inc., Osaka, Japan) performed data collection, management, monitoring, audits, and statistical analyses.

Patients

Patients who were SARS-CoV-2-positive with mild COVID-19 and could stay at isolation facilities in Nagasaki city, Nagasaki, Japan, were eligible for this study. The main inclusion criteria were as follows: (1) presence of SARS-CoV-2 [a positive SARS-CoV-2 antigen test or polymerase chain reaction (PCR) test], (2) arterial oxygen saturation (SpO₂) ≥ 96%, and (3) age of 20–65 years. The main exclusion criteria were: (1) obesity [body mass index (BMI) ≥ 30 kg/m²], (2) presence of severe dyspnea, chest pain, or hemosputum, (3) history of COVID-19, (4) treatment or planned treatment with neutralizing antibody drugs for SARS-CoV-2, (5) treatment with immunosuppressive agents, antirheumatic agents, corticosteroids, or immunoglobulins, or (6) administration of oral probiotics. These eligibility criteria were assessed and judged at the participating medical institutions through testing and medical interview by the participating physicians. The detailed eligibility criteria are described in our protocol [20].

Under Japan's legal regulations and the Ministry of Health, Labour and Welfare (MHLW) guidelines at the time of the present study, patients with fever or symptoms suggestive of COVID-19 were required to undergo testing at a nearby primary care or public institution. If they tested positive for SARS-CoV-2, they were mandated—under provisions equivalent to Category II infectious diseases—to receive further evaluation and treatment at a designated specialized medical institution to be admitted to an isolation facility. Accordingly, patients who tested positive at their primary care or public institution and subsequently visited one of the seven participating medical institutions were enrolled in this study after providing written informed consent. All enrolled patients were admitted to designated isolation facilities. Subsequently, with the emergence of the Omicron variant, even patients with mild or asymptomatic infection were required to undergo inpatient isolation. Therefore, the study protocol was amended to allow the enrollment of hospitalized patients.

Randomization, Study Intervention, and Observation

Eligible participants were randomly assigned to the LC-Plasma or placebo group in approximately a 1:1 ratio, with a minimization procedure to balance allocation factors (age: < 50 years or ≥ 50 years; SARS-CoV-2 vaccination status; use of anti-SARS-CoV-2 agents). The patients assigned to the LC-Plasma or placebo group were asked to consume LC-Plasma-containing capsules (200 mg/day, containing heat-killed LC-Plasma of at least 4.0 × 10¹¹ cells) or placebo capsules (200 mg/day of maltodextrin instead of LC-Plasma), respectively, for 14 days. All patients were subsequently observed for 14 days (observation points: days 1, 4, 8, and 14). Detailed interventions and observations have been described previously [20].

Study Endpoints

The primary endpoint of this study was the change in subjective symptoms measured using the severity score [21] and the visual analog scale (VAS). Subjective symptoms (fatigue, anorexia, headache, cough, shortness of breath, chest pain, smell disturbance, and taste disturbance) were assessed using a 4-point Likert scale (not affected: 0 points; little effect: 1 point; affected: 2 points; and severely affected: 3 points) through a questionnaire completed by the enrolled participants by themselves. The total severity score was calculated as the sum of the scores for these eight symptoms. Secondary endpoints included nasopharyngeal viral load of SARS-CoV-2, pDC and its activation markers, SARS-CoV-2-specific antibodies, serum cytokines, FN or IFN-inducible antiviral effectors, and the proportion of patients who visited the emergency room or were hospitalized during the study period.

SARS-CoV-2 Detection

Quantitative RT-PCR targeting the N2 protein was performed after nucleic acid extraction from nasopharyngeal swabs. PCR was conducted using the Thunderbird Probe One-Step qRT-PCR kit (TOYOBO) and QuantStudio 6 Pro Real-Time PCR System (Thermo Fisher Scientific). The viral load was expressed as copies per 5 μL.

Preparation and Analysis of Peripheral Blood Mononuclear Cell

Peripheral blood mononuclear cells (PBMCs) were collected in 8-mL BD Vacutainer CPT tubes (Becton Dickinson). The tubes were subsequently centrifuged for 15 min at 1800g at room temperature. The layers containing PBMCs were collected and washed with 10 mL of PBS (GIBCO). After centrifugation for 15 min at 300g, the cell pellets were resuspended in 3 mL of CELLBANKER 1 plus (ZENOAQ). The suspended PBMCs were divided into three aliquots and were stored at – 196℃ until analysis.

During the analysis, PBMCs were stained with a fluorescent dye conjugated to Abs: FITC-Lineage cocktail 1, 7-amino-actinomycin D (7-AAD) (BD Pharmingen), PE-CD123 (Clone: AC145), APC -BDCA4 (Clone: AD5-17F6) (Miltenyi Biotec), BV421-CD86 (Clone: IT2.2), and BV510-HLA-DR (Clone: L243) (BioLegend). The cells were washed with FACS buffer (0.5% BSA in PBS) and resuspended in the same buffer for FACS analysis. Data were collected using an Attune Flow Cytometer (Thermo Fisher Scientific) and analyzed using the FlowJo software (Tree Star). pDCs were defined as Lineage-CD123⁺CD304 (BDCA4)⁺ double-positive cells. Cells stained with 7-AAD were denoted as dead cells and excluded from the analysis.

Serum Cytokines Measurement

Serum concentrations of MCP-1 and IL-6 were determined using the Human CCL2/MCP-1 Quantikine ELISA Kit (R&D Systems) and the Human IL-6 CLEIA Kit (Fujirebio), respectively.

qRT-PCR of Interferon-Stimulated Genes

Total RNA and cDNA were extracted from PBMCs using the RNeasy Mini Kit (Qiagen) and iScript cDNA synthesis kit (Bio-Rad), respectively, according to the manufacturer’s protocols. qRT-PCR was performed using SYBR Premix Ex Taq (TaKaRa Bio) on a LightCycler 480 (Roche). Actb was used as a reference gene. Primers for Actb and interferon-stimulated genes are shown in Table S1.

Sample Size

We set the target sample size at 100 patients, with 50 patients in each group, based on the sample size calculation using data from the previously reported case series that used the severity score [21]. The rationale for the sample size calculation is described in our protocol [20].

Statistical Analysis

All tests were two-sided, and a p value < 0.05 was considered statistically significant. As this was an exploratory trial, multiplicity was not adjusted for all endpoints. A statistical analysis plan was developed before the database lock.

Three analysis sets were defined in this study, and the full analysis set (FAS) included all registered patients. Patients with severe protocol violations, such as registration without consent or registration outside the enrollment period, were excluded from the FAS. The per-protocol set (PPS) excluded patients with protocol violations, such as violation of eligibility criteria, use of prohibited or restricted concomitant treatments, or poor adherence to test capsules (less than 75% or more than 120%). The safety analysis set (SAS) included all patients registered in this study who received at least one dose of the test capsules.

The primary endpoint of this study, the change in subjective symptoms measured by the severity score [21], was calculated by summarizing the severity scores of the eight symptoms. Summary statistics of the change in the total severity score from baseline to day 14 were calculated. Analysis of covariance (ANCOVA) was conducted to test the null hypothesis that the change in the total severity score from baseline to day 14 was the same in both groups. The allocation factors were used as covariates in the ANCOVA. For the secondary endpoints, summary statistics (number of patients, mean, standard deviation, minimum, first quartile, median, third quartile, and maximum) for measurements, changes from baseline, and percentage changes from baseline were calculated for continuous data. Frequencies and proportions were calculated for the categorical data. Detailed statistical analysis methods have been described previously [20].

Results

Baseline Characteristics of Patients

A flowchart of the study population is presented in Fig. 1. A total of 187 patients were evaluated for eligibility, of whom 100 were enrolled and randomized into the LC-Plasma (51 patients) or placebo group (49 patients). One patient was excluded from the placebo group because of consent withdrawal. After the initiation of the study intervention, one patient in the LC-Plasma group and two patients in the placebo group were found to be SARS-CoV-2-negative by PCR and were excluded from the FAS, resulting in an FAS comprising 50 patients in the LC-Plasma group and 46 patients in the placebo group. The baseline patient characteristics are presented in Table 1. There were no significant differences in patient characteristics between the groups. More than 80% of the patients in both groups were vaccinated with SARS-CoV-2 mRNA vaccines two or three times. Four patients in the LC-Plasma group (molnupiravir in three patients and nirmatelvir/ritonavir in one patient) and two patients (molnupiravir) in the placebo group were administered antiviral agents during the course. The duration from COVID-19 onset to enrollment was approximately 4 days in both groups. Approximately 90% of patients in both groups were symptomatic based on the severity score, including eight symptoms [21]. The most frequent symptom at registration was cough, followed by headache, fatigue, and anorexia. One-third and 13–14% of the patients in both groups presented with chest pain and shortness of breath, respectively. Smell and taste disturbances were observed in 12% and 4.4% of patients in the LC-Plasma and placebo groups, respectively.

Fig. 1.

Study flow chart showing patient enrollment, allocation, and analysis. FAS full analysis set, PPS per-protocol set, SAS safety analysis set

Table 1.

Patients’ characteristics

	L. lactis strain plasma	Placebo	Between-group p value
Age (year)	38.1 ± 13.5 (50)	38.7 ± 12.5 (46)	0.83
Female sex [n (%)]	23 (46.0)/50	18 (39.1)/46	0.50
BMI (kg/m2)	23.1 ± 3.3 (50)	23.1 ± 3.0 (46)	0.92
Vaccination of SARS-CoV-2 vaccine	42 (84.0)/50	39 (84.8)/46	0.92
0–1 time	8 (16.0)/50	7 (15.2)/46	0.92
2–3 times	42 (84.0)/50	39 (84.8)/46
Use of antiviral agent	4 (8.0)/50	2 (4.3)/46	0.68a
Onset of COVID-19	50 (100.0)/50	46 (100.0)/46	–
Days from the onset of COVID-19	4.3 ± 1.1 (50)	4.0 ± 1.2 (46)	0.14
Symptomatic patients in severity score	44 (88.0)/50	41 (91.1)/45	0.74a
Cough	38 (76.0)/50	38 (82.6)/46	0.43
Headache	20 (40.0)/50	22 (47.8)/46	0.44
Fatigue	22 (44.0)/50	25 (54.3)/46	0.31
Anorexia	20 (40.0)/50	14 (31.1)/45	0.37
Shortness of breath	15 (30.0)/50	14 (30.4)/46	0.96
Chest pain	7 (14.0)/50	6 (13.3)/45	0.92
Smell disturbance	3 (6.0)/50	2 (4.4)/45	1.00a
Taste disturbance	6 (12.0)/50	3 (6.5)/46	0.49a
Smell or taste disturbance	6 (12.0)/50	2 (4.4)/45	0.27a

Data are presented as the mean ± standard deviation (n) for continuous variables and as the number of patients (%)/n for categorical variables. The Chi-squared test or Fisher’s exact test for categorical variables and the two-sample t test for continuous variables were performed

COVID-19 coronavirus disease 2019, SARS-CoV-2 severe acute respiratory syndrome coronavirus

^aBetween-group comparison was conducted using Fisher’s exact test, as it did not meet the requirements of the Chi-square test

Vital Signs and Laboratory Data

The vital signs and laboratory data at baseline are shown in Table S2. Vital signs, including body temperature, pulse rate, SpO₂, and respiratory rate, were within normal ranges, without between-group differences. Laboratory data were within the reference ranges, except for CRP, which was high, averaging 0.79 mg/dL in both groups.

Primary Endpoint

The primary endpoint of this study was the change in subjective symptoms measured using the severity score [21] and VAS. Changes in the total scores of eight symptoms (fatigue, anorexia, headache, cough, shortness of breath, chest pain, smell disturbance, and taste disturbance) and VAS scores over 14 days are shown in Fig. 2. The total severity scores started at an average of 4.08 and 4.24 on day 1 and decreased to an average of 0.756 and 1.022 on day 14 in the LC-Plasma and placebo groups, respectively (Fig. 2A). Similarly, the total VAS scores averaged 11.174 and 10.193 on day 1 and decreased to 1.591 and 1.247 on day 14 in the LC-Plasma and placebo groups, respectively (Fig. 2B). The adjusted mean changes in total severity and VAS scores from day 1 did not differ between the groups throughout the observation period.

Fig. 2.

Changes in total scores of the severity score and visual analog scale (VAS) for eight symptoms, and the time course and proportion of patients without systemic, respiratory, or olfactory and gustatory symptoms. A, B Data are presented as means ± standard errors. Adjusted mean changes were estimated using analysis of covariance (ANCOVA) with allocation factors (age: < 50 or ≥ 50 years; SARS-CoV-2 vaccination status: vaccinated or unvaccinated; use of anti-SARS-CoV-2 agents: present or absent) and baseline total severity score or VAS as covariates. C Proportion of patients without systemic symptoms (fatigue, anorexia, or headache). D Proportion of patients without respiratory symptoms (cough, shortness of breath, or chest pain). E Proportion of patients without taste or smell disturbances. Between-group comparisons were performed using Chi-square or Fisher’s exact tests, as appropriate. p < 0.05 for between-group comparisons in adjusted mean change from baseline (A, B) and in proportions of patients (C–E). VAS visual analog scale

Severity and VAS scores (Figure S1) for systemic symptoms (sum of fatigue, anorexia, and headache scores, Figure S1A), respiratory symptoms (sum of cough, shortness of breath, and chest pain scores, Figure S1B), and the sum of smell and taste disturbance scores (Figure S1C) showed no differences between the groups in the adjusted mean changes from day 1 throughout the observation period. Similar trends in the VAS time course were observed for severity scores (Figure S1D–F). There were no statistically significant differences between the groups, except that the reduction in VAS scores for respiratory symptoms from day 1 was significantly greater in the placebo group than in the LC-Plasma group on day 3 (median in change from day 1: − 0.4 in the LC-Plasma group and − 1.7 in the placebo group, p = 0.045). The severity scores and VAS scores for each of the eight symptoms are shown in Figures S2 and S3, respectively. Severity and VAS scores were highest on day 1 and decreased with fatigue, headache, shortness of breath, and chest pain. Improvement in anorexia was observed within 8–9 days, while cough persisted in some patients through day 14. Smell and taste disturbances peaked on days 5–6, and then gradually decreased. All symptoms, except for cough, did not show significant differences in adjusted mean changes from day 1 throughout the observation period. The decrease in the VAS score for cough symptoms was significantly greater in the placebo group than in the LC-Plasma group at early time points on days 2 (median in change from day 1: 0.0 in the LC-Plasma group and − 0.8 in the placebo group, p = 0.009) and 3 (median in change from day 1: − 0.3 in the LC-Plasma group and − 1.1 in the placebo group, p = 0.010).

A post hoc analysis that calculated the proportion of patients without positive severity scores revealed no significant differences between the groups for systemic and respiratory symptoms throughout the observation period (Fig. 2C, D). In contrast, the number of patients without smell and taste disturbance decreased by day 6 and increased after day 7 in both groups. Notably, on day 13, the proportion was significantly higher in the LC-Plasma group (97.8%) compared to the placebo group (82.6%) (p = 0.030). This trend also occurred at later time points (days 8–14) (Fig. 2E). The proportion of patients without positive severity scores for fatigue, anorexia, headache, cough, shortness of breath, and chest pain gradually increased from day 1 and reached 88.9–100.0% on day 14, except for cough, which remained at 64.4–71.1% on day 14 (Figure S4A–F). The proportion of patients who had smell (Figure S4G) or taste (Figure S4H) disturbance showed a different trend compared with the other symptoms. In particular, the proportion of patients without smell disorders decreased from day 1 through days 5–6 and then increased thereafter, with a significantly higher proportion observed in the LC-Plasma group compared to the placebo group on day 10 (95.7% vs. 82.2%, p = 0.048).

SARS-CoV-2 Viral Load

The SARS-CoV-2 viral load and percent change in SARS-CoV-2 viral load on days 1, 4, and 8 are shown in Fig. 3A and B, respectively. The SARS-CoV-2 viral load significantly decreased from day 1 to day 8 in both groups {percent change from day 1 [95% confidence interval (95% CI)]: − 72.7% [− 79.8%, − 65.6%], p < 0.001 in the LC-Plasma group and − 71.3% [− 81.0%, − 61.6%], p < 0.001}. However, only the LC-Plasma group showed a significant SARS-CoV-2 viral load decrease from day 1 to day 4 [− 27.9% (− 36.4%, − 19.3%), p < 0.001], whereas the placebo group did not [− 10.7% (− 36.0%, 14.6%), p = 0.40]. The percent change in the SARS-CoV-2 viral load showed a significant decrease from day 1 to day 8 in both groups, whereas only the LC-Plasma group showed a decrease on day 4 (Fig. 3).

Fig. 3.

Changes in SARS-CoV-2 viral load. A SARS-CoV-2 viral load presented as box plots. B Mean ± standard error of percent change from day 1 in log-transformed viral load. Two-sample t tests (between groups) and one-sample t tests (within groups) were conducted using log-transformed values. p < 0.05 for between-group comparison of percent change from day 1. ^†p < 0.05 for within-group comparison of percent change from day 1. SARS-CoV-2 severe acute respiratory syndrome coronavirus 2

pDC Activation

The percentage of peripheral lymphocytes at day 1 was 36–39%, which decreased up to 5% at day 8, with no difference between the groups [between-group difference in change (95% CI): − 1.66% (− 6.28%, 2.96%), p = 0.48] (Fig. 4A). Conversely, although the pDC count (defined as lineage CD123 + CD304 (BDCA4) + double positive cells) was significantly lower in the LC-Plasma group (0.59 ± 0.56/μL) compared to that the placebo group (0.91 ± 0.75/μL) on day 1 [between-group difference (95% CI): − 0.32 (− 0.59, − 0.05), p = 0.021], it showed a numerical increase by days 4 and 8 in the LC-Plasma group, whereas it decreased over the same period in the placebo group (Fig. 4B). After the adjustment, which included values at baseline using a mixed-effects model for repeated measures, pDC counts increased nearly 48% on day 8 in the LC-Plasma group (adjusted percent change from day 1: 48.0 ± 28.0%). This increase was significant (p = 0.0498) compared with that in the placebo group (adjusted percent change from day 1: − 1.8 ± 28.3%) (Fig. 4C). The proportion of pDC among lineage-negative cells (CD3⁻, CD16⁻, CD19⁻, CD20⁻, CD14⁻, CD56⁻) continuously decreased through day 8 in the placebo group to nearly − 30%, whereas the LC-Plasma group kept a higher abundance of pDC (adjusted percent change at day 8 from day 1, − 3.3 ± 15.8% and − 29.8 ± 16.0%, respectively, p = 0.042) (Fig. 4D). Among the pDC activation markers (HLA-DR and CD86), HLA-DR expression increased from day 1 to day 4 in the LC-Plasma group and from day 1 to days 4 and 8 in the placebo group; however, the adjusted percent changes did not differ significantly between the groups (Fig. 4E, F).

Fig. 4.

Peripheral lymphocytes, plasmacytoid dendritic cells (pDCs), and pDC activation markers. Data are presented as means ± standard errors. One-sample t tests assessed within-group changes (A). Two-sample t tests compared measurement values between groups (B). A mixed-effects model for repeated measures with an unstructured covariance structure was used for between-group comparisons of changes from baseline in C, D, including treatment group, time (days), interaction between treatment and time, allocation factors, baseline values as fixed effects, and participants as random effects. p < 0.05 for between-group comparisons of measurement values (B) and adjusted percent change from day 1 (C, D). ^†p < 0.05 for within-group changes from day 1 (A, E, F) and percent changes from day 1 (C, D). pDCs were defined as Lineage-CD123⁺CD304 (BDCA4)⁺ double-positive cells. MFI median fluorescent intensity

Immunoglobulins, Cytokines, and Interferon-Inducible Genes

Serum anti-SARS-CoV-2 IgM and IgG levels increased on days 4 and 8, without between-group differences (Figure S5). The serum interleukin-6 and MCP-1 levels decreased on days 4 and 8, without between-group differences (Figure S6). The relative expression mRNA levels of interferon-inducible genes, such as ISG15, RSAD2, OAS1, MxA, IRF9, and IFIT1, decreased from day 1 to day 8 in both groups, following a declining trend with no significant differences between groups (Figure S7). Change in relative expression levels of IFN-α were significantly higher in the placebo group (0.05 ± 0.06) compared with those in the LC-Plasma group (− 0.05 ± 0.05) on day 4 [adjusted between-group difference in change (95% CI): − 0.10 (− 0.20, 0.00), p = 0.040]. IFN-β did not show significant change in either groups, without between-group differences. The expression levels of IFN-λ were not detectable in all patients and observation points (data not shown).

Hospital Visit Or Hospitalization During the Course

Under Japan's legal regulations and MHLW guidelines at the time of the present study, patients who tested positive for SARS-CoV-2 were required to be admitted to isolation facilities. Accordingly, all patients in this study stayed in designated isolation facilities. If any adverse events occurred that required medical treatment, the patients were to be transferred to one of the medical institutions participating in this study. No patients in either group were hospitalized during the observation period. One patient (2.0%) in the LC-Plasma group and one patient (2.2%) in the placebo group visited the hospital because of a persistent cough, both occurring after discharge from the designated isolation facilities on day 8. No differences were observed between the groups (data not shown).

Safety Assessment

During the study period, adverse events occurred in three patients (5.9%) in the LC-Plasma group and one patient (2.1%) in the placebo group. Diarrhea (2.0%), cough-variant asthma (2.0%), and urticaria (2.0%) were observed in one patient each in the LC-Plasma group, while one patient in the placebo group experienced a rib fracture (2.1%). No serious adverse events were reported in either group, and the frequency of adverse events did not differ between the groups.

Discussion

This study investigated the efficacy of LC-Plasma in attenuating symptoms in patients with mild COVID-19 during the SARS-CoV-2 Omicron BA.1 pandemic [22]. This is the first trial to administer LC-Plasma to patients with viral infections. The primary outcomes did not show an advantage of LC-Plasma in improving the total score of eight subjective symptoms measured by the severity score [21] and VAS. However, a post hoc analysis revealed that the proportion of patients without smell and taste disturbances was significantly higher in the LC-Plasma group than in the placebo group at later time points. This suggests the potential efficacy of LC-Plasma in alleviating COVID-19-related symptoms in the upper airways, which were the only symptoms included in this study. The study was originally planned during the SARS-CoV-2 Delta variant epidemic, characterized by more severe lower respiratory tract symptoms, which may have influenced the lack of significant improvement in the primary endpoint.

Our findings suggest that LC-Plasma is effective for early SARS-CoV-2 viral reduction in COVID-19, supported by evidence of pDC activation in the LC-Plasma group. LC-Plasma intake increased the peripheral number of pDCs and maintained their percentage. To our knowledge, this is the first report of a non-pharmaceutical supplement or food activating and maintaining pDC levels in COVID-19 patients. In severe COVID-19, pDCs are severely depleted and remain low for extended periods, a phenomenon termed the “pDC desert” [23]. Activation of pDCs by LC-Plasma may contribute to early viral clearance and improvement of subjective upper airway symptoms. Although the mechanisms underlying smell and taste loss in COVID-19 are not fully understood, possible causes include direct viral damage to nerves, olfactory cells, and taste buds, as well as secondary neurological damage caused by inflammation [24]. LC-Plasma may suppress or attenuate such neurological damage through pDC activation, thereby facilitating the immune response. The lack of detected effects on type I IFN or IFN-stimulated genes in this study may be attributed to the timing of sample collection; these genes peak early after onset, especially in mild cases, and rapidly return to baseline [25]. As the average time from onset to enrollment was over 4 days, and by day 4 post-enrollment approximately 7–8 days had elapsed since symptom onset, upregulation of IFN-stimulated genes may have been missed.

Several antiviral agents have been used to treat COVID-19; however, few have proven effective against the Omicron variant infection in patients without risk factors or those vaccinated with SARS-CoV-2 mRNA vaccines [26]. Ensitrelvir fumaric acid has been approved for COVID-19 treatment because it shortens the duration of five symptoms (fatigue, fever, nasal congestion, sore throat, and cough) and effectively reduces viral load [27]. Combining antivirals with LC-Plasma may accelerate SARS-CoV-2 clearance and improve COVID-19 symptoms. Further studies are needed to verify potential therapeutic strategies combining antivirals and supplements.

LC-Plasma is a food supplement, not a medical agent, and its safety has been confirmed. Previous studies demonstrated that intake of 250 mg/day LC-Plasma-containing food (with at least 5.0 × 10¹¹ heat-killed LC-Plasma cells) for 4 weeks caused no adverse events [28]. LC-Plasma-containing beverages, yogurt, and supplements with at least 1.0 × 10¹¹ cells have been commercially available since 2012 without reported health problems. In this study, no safety concerns arose after 14 days of administering 200 mg/day LC-Plasma-containing food (containing at least 4.0 × 10¹¹ heat-killed cells), which is four times the amount in commercial products, in patients with mild COVID-19. This is the first report demonstrating LC-Plasma’s safety during a human viral infection. Therefore, LC-Plasma may be especially suitable for use in high-risk elderly patients or those with comorbidities.

This study has several limitations. First, it recruited only low-risk patients, none of whom were elderly, and no hospitalizations occurred, limiting the ability to assess LC-Plasma’s effect on disease progression. Further studies are required to assess the availability of LC-Plasma in patients at risk or in more diverse populations, such as older adults, pregnant or lactating women, patients receiving multiple medications (polypharmacy), individuals with contraindications to vaccination, and those who decline vaccination. Second, all participants were Japanese, limiting the generalizability of results to other ethnicities. Third, with an average of over 4 days since onset at enrollment, evaluation of molecular changes, including secondary immune responses, occurring early under infection was limited. Fourth, symptom evaluation was limited to 14 days, which may be insufficient to assess long-term sequelae. Notably, COVID-19 can be accompanied by medium- to long-term symptoms—so-called “long COVID” [4]—with smell and taste disturbances being typical and important indicators reflecting disease severity [5]. Further long-term trials are needed to investigate LC-Plasma’s efficacy for long-COVID symptoms. Fifth, this study did not plan and perform any disaggregated analysis by sex and/or gender, due to lack of evidence regarding sex/gender differences in the efficacy and safety of LC-Plasma. Further studies or analyses are required to assess the sex/gender differences in the efficacy and safety of LC-Plasma in patients with COVID-19.

Conclusion

The intake of LC-Plasma in patients with mild COVID-19-activated pDC decreased SARS-CoV-2 viral load earlier. The study also suggested that the intake of LC-Plasma improved smell and taste disorders more quickly. LC-Plasma is safe and could be a reasonable and easily accessible tool for the treatment of mild COVID-19.

Supplementary Information

Below is the link to the electronic supplementary material.

Author Contribution

Conceptualization: (lead) Kazuko Yamamoto, (equal) Kenta Jounai, Ryohei Tsuji, Daisuke Fujiwara, Katsunori Yanagihara, Koichi Izumikawa, Hiroshi Mukae. Investigation: (lead) Kazuko Yamamoto, (equal) Kazuko Yamamoto, Tsuyoshi Inoue, Takaya Ikeda, Toyomitsu Sawai, Yosuke Nagayoshi, Koji Hashiguchi, Yoji Futsuki, Yuichi Matsubara, Yousuke Harada, Nobuyuki Ashizawa, Susumu Fukahori, Naoki Iwanaga, Takahiro Takazono, Takashi Kido, Hiroshi Ishimoto, Naoki Hosogaya, Noriho Sakamoto, Masato Tashiro, Takeshi Tanaka, Chizu Fukushima, Kenji Ota, Kosuke Kosai, Akitsugu Furumoto, Katsunori Yanagihara, Koichi Izumikawa, Hiroshi Mukae. Resources: (lead) Kenta Jounai, (equal) Ryohei Tsuji, Daisuke Fujiwara. Writing—original draft: (lead) Kazuko Yamamoto, (equal) Kenta Jounai. Writing—review and editing: (lead) Kazuko Yamamoto, (equal) Tsuyoshi Inoue, Takaya Ikeda, Toyomitsu Sawai, Yosuke Nagayoshi, Koji Hashiguchi, Yoji Futsuki, Yuichi Matsubara, Yousuke Harada, Nobuyuki Ashizawa, Susumu Fukahori, Naoki Iwanaga, Takahiro Takazono, Takashi Kido, Hiroshi Ishimoto, Naoki Hosogaya, Noriho Sakamoto, Masato Tashiro, Takeshi Tanaka, Chizu Fukushima, Kenta Jounai, Ryohei Tsuji, Daisuke Fujiwara, Kenji Ota, Kosuke Kosai, Akitsugu Furumoto, Katsunori Yanagihara, Koichi Izumikawa, Hiroshi Mukae. Visualization: (lead) Kazuko Yamamoto, (equal) Kenta Jounai, Ryohei Tsuji, Daisuke Fujiwara. Supervision: (lead) Kazuko Yamamoto, (equal) Hiroshi Mukae. Project administration: (lead) Kazuko Yamamoto, (equal) Hiroshi Mukae. Funding acquisition: (lead) Kazuko Yamamoto, (equal) Hiroshi Mukae.

Funding

This study was funded by the Kirin Holdings Co., Ltd. (Grant number: H21003539). Kirin Holdings Co., Ltd. contributed to the conception and design of the study, preparation of test capsules, and analysis of immune cells in a blinded manner as the core laboratory. Kirin Holdings Co., Ltd. did not play any role in patient enrollment, random assignment, intervention, observation, data management, or statistical analysis. The grant from Kirin Holdings Co., Ltd. also covered the fee for the medical writing support and journal’s rapid service.

Data Availability

Datasets generated and analyzed during this study are not publicly available because of the absence of a statement in the study protocol and informed consent documents, enabling data sharing with a third party after the end of the study. Public data sharing was not approved by certified review boards. The datasets will be shared with interested researchers upon request (kazukomd@med.u-ryukyu.ac.jp). However, the provision of the datasets may take a while because following steps may be required prior to the data provision; development of new protocol, approval from appropriate ethical review board, legally-required informed consent procedure (probably opt-out) in Japan, and data share contract or material transfer agreement.

Declarations

Conflict of Interest

Kazuko Yamamoto received research funds from Kirin Holdings Co., Ltd. for this study and another research grant from Fisher & Paykel Healthcare Ltd. Tsuyoshi Inoue received research funds from Kirin Holdings Co., Ltd. for this study. It belonged to a donated course from Kyowa Kirin Co., Ltd. Kenta Jounai, Ryohei Tsuji, and Daisuke Fujiwara are employees of Kirin Holdings Co. Ltd. Koichi Izumikawa received a research grant for this study from Kirin Holdings Co., Ltd. Hiroshi Mukae received a research grant from Taisho Pharmaceutical Co. Ltd. All funding agencies, except Kirin Holdings Co., Ltd., played no role in the study design, data collection and analysis, decision to publish, or manuscript preparation. Takaya Ikeda, Toyomitsu Sawai, Yosuke Nagayoshi, Koji Hashiguchi, Yoji Futsuki, Yuichi Matsubara, Yosuke Harada, Nobuyuki Ashizawa, Susumu Fukahori, Naoki Iwanaga, Takahiro Takazono, Takashi Kido, Hiroshi Ishimoto, Naoki Hosogaya, Noriho Sakamoto, Masato Tashiro, Takeshi Tanaka, Chizu Fukushima, Kenji Ota, Kosuke Kosai, Akitsugu Furumoto, and Katsunori Yanagihara declare no conflicts of interest.

Ethical Approval

The study protocols were inspected and approved (approval no. CRB21-009) in November 2021 by the Clinical Research Review Board of Nagasaki University. This study was registered with the Japan Registry of Clinical Trials (jRCT) (registration number: jRCTs071210097) in December 2021. The trial protocol has been described previously [20]. Patient enrollment was conducted from December 2021 to April 2022. The study was conducted in accordance with the Declaration of Helsinki, Clinical Trials Act, and other current legal regulations in Japan. Written informed consent was obtained from all enrolled patients who met the eligibility criteria before the intervention. To avoid bias and ensure quality, a third-party entity (Soiken, Inc., Osaka, Japan) performed data collection, management, monitoring, audits, and statistical analyses.