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

Abstract

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.

FIGURE 1

(A) Mechanisms through which topically applied beneficial lactobacilli can act against respiratory viral disease, and (B) Rationale for selection of Lactobacillaceae strains for topical application against respiratory viral disease. The modes of action experimentally explored in this study are indicated in bold frames. AECs, airway epithelial cells; APC, antigen‐presenting cell; IFN, interferon; Teff, T effector cell (based on Spacova et al., 2021).

FIGURE 2

(A) Stimulation of interferon regulatory factors (IRFs) and (B) nuclear factor (NF)‐κB by selected Lactobacillaceae strains in human monocytes, and (C–F) L. casei AMBR2, L. rhamnosus GG, L. plantarum WCFS1, and their combination AMBR2/WCFS1/LGG activate immune pathways involved in antiviral responses. Live L. casei AMBR2, L. rhamnosus GG and L. plantarum WCFS1 and their combination induce (C) nuclear factor (NF)‐κB and (D) interferon regulatory factors (IRFs) in human THP1‐Dual monocytes upon co‐incubation. UV‐inactivated L. rhamnosus GG and L. plantarum WCFS1 and their combination with L. casei AMBR2 also induce (E) NF‐κB and (F) IRFs in human THP1‐Dual monocytes. The medium condition represents the cells as such and serves as a baseline, while Poly(I:C) at 50 μg/ml with Lipofectamine (Poly(I:C)50) serves as the control IRF inducer and LPS (lipopolysaccharide) at 20 ng/ml (LPS20) and LPS‐producing E. coli DH5α serve as the control NF‐κB inducer. Data are depicted as mean ± SD per condition. *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001 as determined by a One‐way ANOVA test followed by Dunnett's multiple comparisons test compared to the medium condition.

FIGURE 3

L. casei AMBR2, L. rhamnosus GG, L. plantarum WCFS1, and their combination AMBR2/WCFS1/LGG decrease cytopathogenic effects of HCoV‐229E, RSV, and influenza A/H1N1, A/H3N2 and B viruses in vitro. L. casei AMBR2, L. rhamnosus GG, and L. plantarum WCFS1 that were added together with the HCoV‐229E virus (A) or pre‐incubated with HCoV‐229E (B), RSV (C), influenza A/H1N1 (D), A/H3N2 (E) or influenza B (F) could inhibit the cytopathic effects induced in human cells. Phosphate‐buffered saline (PBS) serves as the control that does not affect viral infection, E. coli DH5α represents a control laboratory strain. Data depicted as mean ± SD per condition. *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001 as determined by a One‐way ANOVA test followed by Dunnett's multiple comparisons test compared to the medium or PBS conditions. CFU, colony‐forming units; EC50, 50% Effective concentration producing 50% inhibition of virus‐induced cytopathic effects, as determined by measuring the cell viability with the colorimetric MTS cell viability assay.

FIGURE 4

Viability of individual and combined L. casei AMBR2, L. plantarum WCFS1, and L. rhamnosus GG in (A, B) powder or (C) the throat spray formulation; Immunostimulatory activity of the powders (D, E) and the placebo and verum spray formulation without or with lactobacilli, respectively (F, G). The viability of the strain combination was evaluated at different storage temperatures over time; 4, 15, and 25°C. CFU, colony‐forming units. The medium condition represents the cells as such and serves as a baseline, Poly(I:C) at 50 μg/ml with Lipofectamine (Poly(I:C)50) serves as control IRF inducer (expressed as luminescence units) and LPS at 20 ng/ml (LPS20) serves as control NF‐κB inducer (expressed as SEAP activity units). Data are depicted as mean ± SD per condition. *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001 as determined by a One‐way ANOVA test followed by Dunnett's multiple comparisons test compared to the medium condition (dotted line).

FIGURE 5

Evaluation of lactobacilli retention within the throat microbiome of healthy volunteers after spray application. (A) Study set‐up; (B) PCoA plot of throat microbiome data based on microbiome analysis via 16S rRNA amplicon sequencing; (C) Relative abundance of the administered lactobacilli in throat swabs based on microbiome analysis via 16S rRNA amplicon sequencing; (D) Relative abundance of the administered lactobacilli in throat swabs based on qPCR analysis. Throat swabs were collected at baseline/start (T0), 30 min (T1), and 2 h (T2) after the throat spray was used. The presence of L. casei AMBR2, L. rhamnosus GG, and L. plantarum WCFS1 was evaluated via 16S rRNA amplicon sequencing (relative abundances) in panel C. At 30 min and 2 h, and qPCR with species‐specific primers was used to estimate the CFU/ml counts in the verum group in panel D. Based on the standard curve, the detection limit was estimated to be at 10³ CFU/ml.