Development of a live biotherapeutic throat spray with lactobacilli targeting respiratory viral infections.

Respiratory viruses such as influenza viruses, respiratory syncytial virus (RSV), and coronaviruses initiate infection at the mucosal surfaces of the upper respiratory tract (URT), where the resident respiratory microbiome has an important gatekeeper function. In contrast to gut-targeting administration of beneficial bacteria against respiratory viral disease, topical URT administration of probiotics is currently underexplored, especially for the prevention and/or treatment of viral infections. Here, we report the formulation of a throat spray with live lactobacilli exhibiting several in vitro mechanisms of action against respiratory viral infections, including induction of interferon regulatory pathways and direct inhibition of respiratory viruses. Rational selection of Lactobacillaceae strains was based on previously documented beneficial properties, up-scaling and industrial production characteristics, clinical safety parameters, and potential antiviral and immunostimulatory efficacy in the URT demonstrated in this study. Using a three-step selection strategy, three strains were selected and further tested in vitro antiviral assays and in formulations: Lacticaseibacillus casei AMBR2 as a promising endogenous candidate URT probiotic with previously reported barrier-enhancing and anti-pathogenic properties and the two well-studied model strains Lacticaseibacillus rhamnosus GG and Lactiplantibacillus plantarum WCFS1 that display immunomodulatory capacities. The three strains and their combination significantly reduced the cytopathogenic effects of RSV, influenza A/H1N1 and B viruses, and HCoV-229E coronavirus in co-culture models with bacteria, virus, and host cells. Subsequently, these strains were formulated in a throat spray and human monocytes were employed to confirm the formulation process did not reduce the interferon regulatory pathway-inducing capacity. Administration of the throat spray in healthy volunteers revealed that the lactobacilli were capable of temporary colonization of the throat in a metabolically active form. Thus, the developed spray with live lactobacilli will be further explored in the clinic as a potential broad-acting live biotherapeutic strategy against respiratory viral diseases.

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.

Selective targeting of skin pathobionts and inflammation with topically applied lactobacilli.

Tailored skin microbiome modulation approaches with probiotics are highly challenging. Here, we show that lactobacilli are underestimated members of the skin microbiota. We select specific strains of nomadic lactobacilli for their functional applicability on the skin and capacity to inhibit growth and inflammation by skin pathobionts. The strains are formulated as microcapsules for topical formulations and tested in patients with mild-to-moderate acne. The selected lactobacilli are able to reduce inflammatory lesions in a pilot and placebo-controlled study. Daily application for 8 weeks is associated with an in vivo temporary modulation of the microbiome, including a reduction in relative abundance of staphylococci and Cutibacterium acnes, and an increase in lactobacilli. The reduction in inflammatory lesions is still apparent 4 weeks after the topical application of the lactobacilli ended, indicating a possible additional immunomodulatory effect. This study shows that carefully selected and formulated lactobacilli are a viable therapeutic option for common acne lesions.

The use of 3 selected lactobacillary strains in vaginal probiotic gel for the treatment of acute Candida vaginitis: a proof-of-concept study.

In vitro studies suggest that certain probiotic bacterial strains have potential activity against opportunistic infections such as Candida. There are few in vivo trials using probiotics as a single treatment for acute Candida vulvovaginitis (CV). In this open-label, proof-of-concept study, selected Lactobacillus strains were tested in women with acute Candida vaginitis. Twenty women diagnosed with proven, symptomatic CV were instructed to administer a vaginal probiotic gel with L. plantarum YUN-V2.0, L. pentosus YUN-V1.0 and L. rhamnosus YUN-S1.0 for 10 consecutive days. Vaginal rinsing fluid, vaginal culture swab and vaginal smear for fresh wet-mount microscopy were collected before and 7, 14 and 28 days after start of treatment. On average, participating women were 39 years old and had an history of 5 vaginal infections of which 95% was CV. Nine women (45%) completed the study without the need of rescue medication. Women who needed rescue treatment experienced twice as much Candida infections in the past. A negative correlation was found between the clinical composite score and the time to use rescue medication (R2 = 0.127). Seventy-four per cent of participants found the study gel comfortable to use, and 42% of all women would use the tested gel again for this indication. Forty-five per cent of women were treated successfully for acute CV with a novel vaginal gel containing 3 selected Lactobacillus strains. Patients needing rescue treatment were suffering from more severe and long-standing disease. These results warrant for further testing of this new product, especially of its potential in cases with mild to moderate severity, as an adjuvant to antimycotics or as a preventive measure in women with recurrent vulvovaginal candidosis.

Impact of a lactobacilli-containing gel on vulvovaginal candidosis and the vaginal microbiome.

Vulvovaginal candidosis (VVC) is a common condition with severe symptoms and high recurrence rates. Probiotic lactobacilli are explored as alternatives to azole treatments. Although the vaginal microbiota is generally not depleted in lactobacilli during VVC, studies indicate that the functionality and antimicrobial activity of the lactobacilli is impaired. We selected three strains from the Lactobacillus genus complex (L. rhamnosus GG, L. pentosus KCA1 and L. plantarum WCFS1) based on in vitro evaluation and formulated them in a gel for vaginal application. This gel was evaluated in 20 patients suffering from acute VVC, who were followed for four weeks including a 10-day treatment period. The microbiome was assessed through 16S rRNA (bacteria) and internal transcribed spacer (ITS; fungi) amplicon sequencing, supplemented with quantitative PCR, culture and microscopy for Candida evaluation. 45% of women did not require rescue medication (3×200 mg fluconazole), implying an improvement of their symptoms. These women showed similar end concentrations of fungi as women treated with fluconazole. Moreover, fluconazole appeared to reduce numbers of endogenous lactobacilli. Our study points towards important aspects for future selection of lactobacilli for probiotic use in VVC and the need to investigate possible negative influences of azoles on the vaginal bacterial community.

Enhancing the viability of Lactobacillus rhamnosus GG after spray drying and during storage.

Increasing knowledge about the human microbiome has led to a growing awareness of the potential of applying probiotics to improve our health. The pharmaceutical industry shows an emerging interest in pharmaceutical formulations containing these beneficial microbes, the so-called pharmabiotics. An important manufacturing step is the drying of the probiotics, as this can increase the stability and shelf life of the finished pharmabiotic product. Unfortunately, drying also puts stress on microbial cells, thus causing a decrease in viability. We aimed to examine the effect of different drying media and protective excipients on the viability of the prototype probiotic strain Lactobacillus rhamnosus GG after spray drying and during subsequent storage for 28 weeks. The presence of phosphates in the drying medium showed to have a superior protective effect, especially during long-term storage at room temperature. Addition of lactose or trehalose resulted in significantly improved survival rates after drying as well as during long-term storage for the tested excipients. Both disaccharides are characterized by a high glass transition temperature. Maltodextrin showed less protective capacities compared to lactose and trehalose in all tested conditions. The usage of mannitol or dextran resulted in sticky powders and low yields, so further testing was not possible. In addition to optimizing the viability, future research will also explore the functionality of cellular probiotic components after spray drying in order to safeguard the probiotic activity of the formulated pharmabiotics.