Evaluation of the immunogenicity of auxotrophic Lactobacillus with CRISPR-Cas9D10A system-mediated chromosomal editing to express porcine rotavirus capsid protein VP4.

Porcine rotavirus (PoRV) is an important pathogen, leading to the occurrence of viral diarrhoea . As the infection displays obvious enterotropism, intestinal mucosal immunity is the significant line of defence against pathogen invasion. Moreover, as lactic acid bacteria (LAB) show acid resistance, bile salt resistance and immune regulation, it is of great significance to develop an oral vaccine. Most traditional plasmid delivery vectors use antibiotic genes as selective markers, easily leading to antibiotic accumulation. Therefore, to select a food-grade marker in genetically engineering food-grade microorganisms is vital. Based on the CRISPR-Cas9D10A system, we constructed a stable auxotrophic Lactobacillus paracasei HLJ-27 (Lactobacillus △Alr HLJ-27) strain. In addition, as many plasmids replicate in the host bacteria, resulting in internal gene deletions. In this study,we used a temperature-sensitive gene editing plasmidto insert the VP4 gene into the genome, yielding the insertion mutant strains VP4/△Alr HLJ-27, VP4/△Alr W56, and VP4/W56. This recombinant bacterium efficiently induced secretory immunoglobulin A (SIgA)-based mucosal and immunoglobulin G (IgG)-based humoral immune responses. These oral mucosal vaccines have the potential to act as an alternative to the application of antibiotics in the future and induce efficient immune responses against PEDV infection.

Prevention of recurrence of bacterial vaginosis using lactobacilli-containing vaginal tablets among women with HIV: a randomized, placebo-controlled, double-blinded phase IV trial.

OBJECTIVES: The effectiveness of lactobacilli-containing vaginal tablets (VT) in bacterial vaginosis (BV) recurrence prevention among women infected with HIV treated with standard oral metronidazole in Pune, India was studied. METHODS: Women infected with HIV with confirmed BV diagnosis (Nugent score ≥7 and Amsel criteria >3) were enrolled in a 12-month, double-blind, randomized, placebo-controlled, phase IV study between 2018 and 2021. After a standard course of oral metronidazole for 7 days (400 mg three times a day), women were randomly assigned to either lactobacilli-containing or placebo VT arms to receive VTs for 4 months. BV recurrence was assessed after the initial cure from BV. RESULTS: Of the 464 women infected with HIV, 80 women with confirmed BV were enrolled. The retention was affected due to the COVID-19 pandemic (6-month retention rates 78%). The cure was seen in 85% and 93.5% of participants from the treatment and placebo arms, respectively, after four VT cycles. BV recurrence was seen in 41.4% and 44.8% in the treatment and placebo arm, respectively, with no significant difference in the two groups. CONCLUSION: The lactobacilli-containing VT was acceptable and safe; however, the addition of VT over standard oral metronidazole did not show any additional benefit in the prevention of BV recurrence in women infected with HIV, indicating the need for long-term randomized trials among them. Registered at Clinical Trials Registry- India, (CTRI) Number: CTRI/2018/04/013298.

Delivery of antigen to porcine dendritic cells by fusing antigen with porcine dendritic cells targeting peptide.

Dendritic cells (DCs) are professional antigen-presenting cells that can recognize, capture, and process antigens. Fusing molecules targeting DCs with antigens can effectively improve the efficiency with which antigens are recognized and captured by DCs. This targeting strategy can be used for vaccine development to effectively improve the efficiency of antigen recognition and capture by DCs. The targeting sequence of porcine cytotoxic T-lymphocyte associated protein 4 (CTLA4), which binds porcine DCs, was identified in this study. Recombinant Lactobacillus reuteri (L. reuteri) expressing CTLA4-6aa (LYPPPY) and CTLA4-87aa fused to the porcine epidemic diarrhea virus (PEDV) protective antigen core neutralizing epitope (COE) were used to evaluate the ability of the two targeting motifs to bind the B7 molecule on DCs. Our results demonstrate that CTLA4-6aa could bind porcine DCs, and recombinant Lactobacillus expressing the CTLA4-6aa captured by porcine DCs was more efficient than those expressing CTLA4-87aa. In addition, the expression of DC markers, toll-like receptors, and cytokines was significantly higher in the 6aa-COE/L. reuteri-stimulated porcine DCs compared to DCs treated with 87aa-COE/L. reuteri (p<0.01) and recombinant Lactobacillus expressing CTLA4-6aa enhanced the ability of porcine DCs to activate T-cell proliferation. Our analysis of the protein structure revealed that CTLA4-87aa contains intramolecular hydrogen bonds, which may have weakened the intermolecular force between the residues on porcine CTLA4 and that on B7. In conclusion, recombinant Lactobacillus expressing CTLA4-6aa were more efficiently captured by porcine DCs and had a stronger ability to promote DC maturation and enhance T-cell proliferation. The LYPPPY motif is the optimal sequence for binding to porcine DCs. Piglets immunized with recombinant Lactobacillus showed that recombinant Lactobacillus expressing CTLA4-6aa induced significant levels of anti-PEDV-specific IgG and IgA antibody responses. Our study may promote research on DC-targeting strategies to enhance the effectiveness of porcine vaccines.

Randomized, Double-Blind, Placebo-Controlled Trial of a Throat Spray with Selected Lactobacilli in COVID-19 Outpatients.

Primary care urgently needs treatments for coronavirus disease 2019 (COVID-19) patients because current options are limited, while these patients who do not require hospitalization encompass more than 90% of the people infected with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Here, we evaluated a throat spray containing three Lactobacillaceae strains with broad antiviral properties in a randomized, double-blind, placebo-controlled trial. Before the availability of vaccines, 78 eligible COVID-19 patients were randomized to verum (n = 41) and placebo (n = 37) within 96 h of a positive PCR-based SARS-CoV-2 diagnosis, and a 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 for microbiome, viral load, and antibody analyses. The daily reported symptoms were highly variable, with no added benefit for symptom resolution in the verum group. However, based on 16S V4 amplicon sequencing, the acute symptom score (fever, diarrhea, chills, and muscle pain) was significantly negatively associated with the relative abundance of amplicon sequence variants (ASVs) that included the applied lactobacilli (P < 0.05). Furthermore, specific monitoring of these applied lactobacilli strains showed that they were detectable via quantitative PCR (qPCR) analysis in 82% of the patients in the verum group. At the end of the trial, a trend toward lower test positivity for SARS-CoV-2 was observed for the verum group (2/30; 6.7% positive) than for the placebo group (7/27; 26% positive) (P = 0.07). These data indicate that the throat spray with selected antiviral lactobacilli could have the potential to reduce nasopharyngeal viral loads and acute symptoms but should be applied earlier in the viral infection process and substantiated in larger trials. IMPORTANCE Viral respiratory tract infections result in significant health and economic burdens, as highlighted by the COVID-19 pandemic. Primary care patients represent 90% of those infected with SARS-CoV-2, yet their treatment options are limited to analgesics and antiphlogistics, and few broadly acting antiviral strategies are available. Microbiome or probiotic therapy is a promising emerging treatment option because it is based on the multifactorial action of beneficial bacteria against respiratory viral disease. In this study, an innovative topical throat spray with select beneficial lactobacilli was administered to primary COVID-19 patients. A remote study setup (reducing the burden on hospitals and general practitioners) was successfully implemented using online questionnaires and longitudinal self-sampling. Our results point toward the potential mechanisms of action associated with spray administration at the levels of viral loads and microbiome modulation in the upper respiratory tract and pave the way for future clinical applications of beneficial bacteria against viral diseases.

Lactobacilli reduce recurrences of vaginal candidiasis in pregnant women: a randomized, double-blind, placebo-controlled study.

AIMS: The aim of this study was to investigate the effects of lactobacilli strains in preventing the recurrences of vaginal candidiasis (VC) in 78 pregnant women with VC (lactobacilli, n = 39; placebo, n = 39) and the potential benefits on quality of life. METHODS AND RESULTS: The lactobacilli putative probiotic (SynForU-HerCare; two capsules/day of 9·5 log CFU per capsule) or placebo was administered for 8-weeks in a randomized, double-blind, placebo-controlled study. Subjects were assessed for vaginal and gut health conditions at baseline, week-4 and week-8 via questionnaires. The vulvovaginal symptom questionnaire not only covered aspects pertaining to vulvovaginal symptoms but also the quality of life impacts such as emotional, social and sexual. The administration of lactobacilli reduced symptoms of irritation (P = 0·023) and discharge (P = 0·011) starting week-4 and continued after week-8 (P < 0·05), accompanied by reduced symptoms for burning after week-8 (P = 0·046) as compared to the placebo. Patients consuming lactobacilli also showed reduced concern about symptoms after week-4 (P = 0·010) and continued after week-8 (P = 0·001), accompanied by reduced impairment of daily activities attributed to vulvovaginal symptoms (P = 0·012) and continued after week-8 (P = 0·026). Insignificant differences were observed for sexual impacts between treatment groups. The administration of lactobacilli also reduced recurrences of both emotional and social stress as compared to the placebo at both week-4 and week-8 (P < 0·05). Patients consuming lactobacilli showed higher defecation times per week at week-4 (P = 0·010) and week-8 (P = 0·001) as compared to the placebo group, indicating the potential to reduce risks of pregnancy-induced constipation. CONCLUSIONS: Lactobacilli probiotics are beneficial towards pregnant women, especially in reducing vulvovaginal symptoms and recurrences of VC, accompanied by improved emotional and social distress attributed to VC. SIGNIFICANCE AND IMPACT OF THE STUDY: The study demonstrated the preventive and modulatory roles of lactobacilli strains against VC in pregnant women. Taken altogether, our present data illustrated that lactobacilli probiotics are beneficial towards pregnant women, especially in reducing vulvovaginal symptoms and recurrences of VC, accompanied by improved emotional and social distress attributed to VC, thus could be a potential strategy for the maintenance of vaginal health during pregnancy.

Effects of Loigolactobacillus coryniformis K8 CECT 5711 on the Immune Response of Elderly Subjects to COVID-19 Vaccination: A Randomized Controlled Trial.

Elderly people are particularly vulnerable to COVID-19, with a high risk of developing severe disease and a reduced immune response to the COVID-19 vaccine. A randomized, placebo-controlled, double-blind trial to assess the effect of the consumption of the probiotic Loigolactobacillus coryniformis K8 CECT 5711 on the immune response generated by the COVID-19 vaccine in an elderly population was performed. Two hundred nursing home residents >60 yrs that had not COVID-19 were randomized to receive L. coryniformis K8 or a placebo daily for 3 months. All volunteers received a complete vaccination schedule of a mRNA vaccine, starting the intervention ten days after the first dose. Specific IgG and IgA antibody levels were analyzed 56 days after the end of the immunization process. No differences between the groups were observed in the antibody levels. During the intervention, 19 subjects had COVID-19 (11 receiving K8 vs. 8 receiving placebo, p = 0.457). Subgroup analysis in these patients showed that levels of IgG were significantly higher in those receiving K8 compared to placebo (p = 0.038). Among subjects >85 yrs that did not get COVID-19, administration of K8 tended to increase the IgA levels (p = 0.082). The administration of K8 may enhance the specific immune response against COVID-19 and may improve the COVID-19 vaccine-specific responses in elderly populations.

Lacticaseibacillus paracasei DG enhances the lactoferrin anti-SARS-CoV-2 response in Caco-2 cells.

The novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is causing the ongoing global pandemic of coronavirus disease 2019 (COVID-19), which primarily manifests with respiratory distress and may also lead to symptoms associated with the gastrointestinal tract. Probiotics are living microorganisms that have been shown to confer immune benefits. In this study, we investigated the immunomodulatory effects and anti-SARS-CoV-2 activity of three different Lacticaseibacillus probiotic strains, either alone or in combination with lactoferrin, using the intestinal epithelial Caco-2 cell line. Our results revealed that the Lacticaseibacillus paracasei DG strain significantly induced the expression of genes involved in protective antiviral immunity and prevented the expression of proinflammatory genes triggered by SARS-CoV-2 infection. Moreover, L. paracasei DG significantly inhibited SARS-CoV-2 infection in vitro. L. paracasei DG also positively affected the antiviral immune activity of lactoferrin and significantly augmented its anti-SARS-CoV-2 activity in Caco-2 intestinal epithelial cells. Overall, our work shows that the probiotic strain L. paracasei DG is a promising candidate that exhibits prophylactic potential against SARS-CoV-2 infection.

Genome Features and In Vitro Activity against Influenza A and SARS-CoV-2 Viruses of Six Probiotic Strains.

PURPOSE: The aim of this work was to analyze the complete genome of probiotic bacteria Lactobacillus plantarum 8 RA 3, Lactobacillus fermentum 90 TC-4, Lactobacillus fermentum 39, Bifidobacterium bifidum 791, Bifidobacterium bifidum 1, and Bifidobacterium longum 379 and to test their activity against influenza A and SARS-CoV-2 viruses. METHODS: To confirm the taxonomic affiliation of the bacterial strains, MALDI TOF mass spectrometry and biochemical test systems were used. Whole genome sequencing was performed on the Illumina Inc. MiSeq platform. To determine the antiviral activity, A/Lipetsk/1V/2018 (H1N1 pdm09) (EPI_ISL_332798) and A/common gull/Saratov/1676/2018 (H5N6) (EPI_ISL_336925) influenza viruses and SARS-CoV-2 virus strain Australia/VIC01/2020 (GenBank: MT007544.1) were used. RESULTS: All studied probiotic bacteria are nonpathogenic for humans and do not contain the determinants of transmission-type antibiotic resistance and integrated plasmids. Resistance to antibiotics of different classes is explained by the presence of molecular efflux pumps of the MatE and MFS families. Cultures of L. fermentum 90 TC 4, L. plantarum 8 RA 3, and B. bifidum 791 showed a pronounced activity against influenza A viruses in MDCK cells. Activity against the SARS-CoV-2 virus was demonstrated only by the L. fermentum 90 TC 4 strain in VERO cells. CONCLUSIONS: The studied probiotic bacteria are safe, have antiviral activity, and are of great importance for the prevention of diseases caused by respiratory viruses that can also infect the human intestine.

Effects of select dietary supplements on the prevention and treatment of viral respiratory tract infections: a systematic review of randomized controlled trials.

Introduction: Viral respiratory tract infections (RTIs) have been recognized as a global public health burden. Despite current theories about their effectiveness, the true benefits of dietary supplements on the prevention and treatment of viral RTIs remain elusive, due to contradictory reports. Hence, we aimed to evaluate the effectiveness of dietary supplements on the prevention and treatment of viral RTIs.Areas covered: We systematically searched databases of PubMed, Web of Science, Scopus, and Google Scholar through 4 March 2020, to identify randomized controlled trials that evaluated the effects of consuming selected dietary supplements on the prevention or treatment of viral RTIs.Expert opinion: Thirty-nine randomized controlled trials (n = 16,797 participants) were eligible and included. Namely, vitamin D supplementation appeared to improve viral RTIs across cohorts particulate in those with vitamin D deficiency. Among the evaluated dietary supplements, specific lactobacillus strains were used most commonly with selected prebiotics that showed potentially positive effects on the prevention and treatment of viral RTIs. Further, ginseng extract supplementation may effectively prevent viral RTIs as adjuvant therapy. However, longitudinal research is required to confirm these observations and address the optimal dose, duration, and safety of dietary supplements being publicly recommended.

Probiotics: A potential immunomodulator in COVID-19 infection management.

COVID-19 caused by SARS-CoV-2 is an ongoing global pandemic. SARS-CoV-2 affects the human respiratory tract's epithelial cells, leading to a proinflammatory cytokine storm and chronic lung inflammation. With numerous patients dying daily, a vaccine and specific antiviral drug regimens are being explored. Probiotics are live microorganisms with proven beneficial effects on human health. While probiotics as nutritional supplements are long practiced in different cuisines across various countries, the emerging scientific evidence supports the antiviral and general immune-strengthening health effects of the probiotics. Here, we present an overview of the experimental studies published in the last 10 years that provide a scientific basis for unexplored probiotics as a preventive approach to respiratory viral infections. Based on collated insights from these experimental data, we identify promising microbial strains that may serve as lead prophylactic and immune-boosting probiotics in COVID-19 management.

Respiratory syndrome coronavirus-2 response: Microbiota as lactobacilli could make the difference.

Coronavirus disease 2019 (COVID-19) is caused by respiratory syndrome coronavirus qualified as SARS-CoV-2. Viral penetration requires binding of the viral spike (S) protein to a specific cellular receptor (ACE2) highly expressed in a nasal goblet and ciliated cells. In several countries, the COVID-19 evolution was relatively benign compared to others and despite noncompliance with health recommendations on several occasions. In this overview, we attempt to define the criteria that could explain such a difference. Among these criteria, the specificity of Lactobacillus genus strains, as a part of nasal microbiota, could play a role of a barrier against viral penetration and could strengthen the host's immune system in some populations rather than others. In fact, several studies have shown the role of lactic acid bacteria, including lactobacilli, in the prevention of viral respiratory infections. This could provide important information on a possible mechanism of the virus spreading.

Nutraceuticals as immune-stimulating therapy to fight COVID-19. Combination of elements to improve the efficacy.

COVID-19 pandemic has underlined that unknown viral infections, which jump from animals to humans, can be extremely dangerous. In case of new viruses as SARS-CoV2, available drugs can fail to contrast the virus aggressiveness leading patients to death. Long time is necessary to create a vaccine, but immediate solutions are necessary to stop the mortality COVID-19 related. We have learned that the immune-system is the key to reduce the severity of COVID-19 and, through its modulation, it has been possible saving people's life. In this short communication, we discuss the use of nutraceuticals to modulate and stimulate the immune answer for reducing the severity of COVID-19 symptoms. The nutraceuticals are safe and can be administered to all ages. In addition, combination of natural anti-viral elements and immune-stimulating molecules already successfully tested against others upper-respiratory tract infections-could be efficient against SARS-CoV2. We believe that these natural molecules could really be a valid ally against COVID-19, especially in this moment in which a SARS-CoV2 vaccine is still not available.

Efficacy of orally administered porcine epidemic diarrhea vaccine-loaded hydroxypropyl methylcellulose phthalate microspheres and RANKL-secreting L. lactis.

Here, we examined the efficacy of are combinant subunit antigen-based oral vaccine for preventing porcine epidemic diarrhea virus (PEDV). First, we generated a soluble recombinant partial spike S1 protein (aP2) from PEDV in E. coli and then evaluated the utility of aP2 subunit vaccine-loaded hydroxypropyl methylcellulose phthalate microspheres (HPMCP) and RANKL-secreting L. lactis (LLRANKL) as a candidate oral vaccine in pregnant sows. Pregnant sows were vaccinated twice (with a 2 week interval between doses) at 4 weeks before farrowing. Titers of virus-specific IgA antibodies in colostrum, and neutralizing antibodies in serum, of sows vaccinated with HPMCP (aP2) plus LL RANKL increased significantly at 4 weeks post-first vaccination. Furthermore, the survival rate of newborn suckling piglets delivered by sows vaccinated with HPMCP (aP2) plus LL RANKL was similar to that of piglets delivered by sows vaccinated with a commercial killed porcine epidemic diarrhea virus (PED) vaccine. The South Korean government promotes a PED vaccine program (live-killed-killed) to increase the titers of IgA and IgG antibodies in pregnant sows and prevent PEDV. The oral vaccine strategy described herein, which is based on a safe and efficient recombinant subunit antigen, is an alternative PED vaccination strategy that could replace the traditional strategy, which relies on attenuated live oral vaccines or artificial infection with virulent PEDV.