Nasal-spraying Bacillus spores as an effective symptomatic treatment for children with acute respiratory syncytial virus infection

Dien Minh Tran, Tu Thanh Tran, Thuy Thi Bich Phung, Huyen Thi Bui, Phuc Thanh Thi Nguyen, Tam Thi Vu, Nga Thi Phuong Ngo, Mai Thi Nguyen, Anh Hoa Nguyen, Anh Thi Van Nguyen, Dien Minh Tran, Tu Thanh Tran, Thuy Thi Bich Phung, Huyen Thi Bui, Phuc Thanh Thi Nguyen, Tam Thi Vu, Nga Thi Phuong Ngo, Mai Thi Nguyen, Anh Hoa Nguyen, Anh Thi Van Nguyen

Abstract

Respiratory syncytial virus (RSV) is a leading cause of Acute Respiratory Tract Infections (ARTIs) in young children. However, there is currently no vaccine or treatment available for children. Here, we demonstrated that nasal-spraying probiotics containing 5 billion of Bacillus spores (LiveSpo Navax) is an effective symptomatic treatment in a 6-day randomized controlled clinical study for RSV-infected children (n = 40-46/group). Navax treatment resulted in 1-day faster recovery-time and 10-50% better efficacy in relieving ARTI symptoms. At day 3, RSV load and level of pro-inflammatory cytokines in nasopharyngeal samples was reduced by 630 folds and 2.7-12.7 folds respectively. This showed 53-fold and 1.8-3.6-fold more effective than those in the control-standard of care-group. In summary, nasal-spraying Bacillus spores can rapidly and effectively relieve symptoms of RSV-induced ARTIs while exhibit strong impacts in reducing viral load and inflammation. Our nasal-spraying probiotics may provide a basis for simple-to-use, low-cost, and effective treatment against viral infection in general.

Conflict of interest statement

LiveSpo Navax is produced by LiveSpo Pharma Ltd., in which A.H.N is a founder and scientific director. The remained authors report no declarations of interest.

© 2022. The Author(s).

Figures

Figure 1
Figure 1
Diagram displaying the flow of participants involved in the study. From the clinical database, participants were screened for eligibility. Eligible participants who provided consent were randomized in either Control or Navax group. Measurements took place between baseline (day 0) and day 6 of treatment period. Participants were recruited from August 2020 to July 2021. Clinical and subclinical data collection and analysis were conducted from September 2020 to August 2021.
Figure 2
Figure 2
Average values of recorded changes in breath (A), pulse (B), temperature (C) and pulse oxygen (D) between before and after nasal-spraying with LiveSpo Navax (in diagonal-strip pattern) and 0.9% NaCl physiological saline (in white pattern), across 9 spraying times over 3 days.
Figure 3
Figure 3
Days of treatment (A1–G1) and time-dependent percentage (%) of asymptomatics patients (A2–G2) for observation of seven typical symptoms of ARTIs related to RSV infection in the Control (dashed lines) and Navax (solid lines) groups. More than six days of treatment refers to symptoms that have not improved by day 6. Methods to test distribution was verified by Mann–Whitney test.
Figure 4
Figure 4
Real-time PCR TaqMan probes amplification curves (A, B) specifically for RSV taken from two representative nasopharyngeal samples of Control (Control 1_code N74, Control 2_code N66) and Navax groups (Navax 1_code N58, Navax 2_code N43) at day 0 and 3 of treatment. PC, NC are positive and negative controls of RSV; real-time PCR SYBR Green amplification curves specifically for B. subtilis (C) and B. clausii (D) taken from two representative nasopharyngeal samples of Control (Control 1, 2) and Navax (Navax 1, 2) groups at day 3 of treatment. PC, NC are positive and negative controls of B. subtilis and B. clausii; Reducing-fold levels (2△Ct) of RSV load in nasopharyngeal samples of Control and Navax groups at day 3 compared to day 0 (E). The Mann–Whitney test was used to calculate the median difference in viral load reduction in the two groups; Threshold cycle (Ct) of fluorescent signals for B. subtilis and B. clausii measured in nasopharyngeal samples of Control and Navax groups at day 3 (F).
Figure 5
Figure 5
Pro-inflammatory cytokines levels (pg/mL) in nasopharyngeal samples of Control and Navax groups at day 3 compared to day 0. The Wilcoxon test was used to calculate the median differences in (i) IL-6 (A), IL-8 (B), TNF-α (C) levels at day 0 and day 3 in each group. The Mann–Whitney test was used to compared cytokine concentrations (A–C) and cytokine reducing folds (D) between the two groups. Only samples with measurable cytokine concentrations at day 0 were included in the statistical analysis. 95% CI for median in each group and the median difference between the two groups were shown in (A–C). The significance level of all analyzes was set at the p < 0.05.

References

    1. Collins PL, Murphy BR. Respiratory syncytial virus: Reverse genetics and vaccine strategies. Virology. 2002;296:204–211. doi: 10.1006/viro.2002.1437.
    1. Falsey AR, Hennessey PA, Formica MA, Cox C, Walsh EE. Respiratory syncytial virus infection in elderly and high-risk adults. N. Engl. J. Med. 2005;352:1749–1759. doi: 10.1056/NEJMoa043951.
    1. Griffiths C, Drews SJ, Marchant DJ. Respiratory syncytial virus: Infection, detection, and new options for prevention and treatment. Clin. Microbiol. Rev. 2017;30:277–319. doi: 10.1128/CMR.00010-16.
    1. Nair H, et al. Global burden of acute lower respiratory infections due to respiratory syncytial virus in young children: A systematic review and meta-analysis. Lancet. 2010;375:1545–1555. doi: 10.1016/S0140-6736(10)60206-1.
    1. Pretorius MA, et al. The role of influenza, RSV and other common respiratory viruses in severe acute respiratory infections and influenza-like illness in a population with a high HIV sero-prevalence, South Africa 2012–2015. J. Clin. Virol. 2016;75:21–26. doi: 10.1016/j.jcv.2015.12.004.
    1. Shi T, et al. Global, regional, and national disease burden estimates of acute lower respiratory infections due to respiratory syncytial virus in young children in 2015: A systematic review and modelling study. Lancet. 2017;390:946–958. doi: 10.1016/S0140-6736(17)30938-8.
    1. Brown PM, Schneeberger DL, Piedimonte G. Biomarkers of respiratory syncytial virus (RSV) infection: Specific neutrophil and cytokine levels provide increased accuracy in predicting disease severity. Paediatr. Respir. Rev. 2015;16:232–240.
    1. Kakimoto Y, Seto Y, Ochiai E, Satoh F, Osawa M. Cytokine elevation in sudden death with respiratory syncytial virus: A case report of 2 children. Pediatrics. 2016 doi: 10.1542/peds.2016-1293.
    1. Christiaansen AF, et al. Altered Treg and cytokine responses in RSV-infected infants. Pediatr. Res. 2016;80:702–709. doi: 10.1038/pr.2016.130.
    1. Vazquez Y, et al. Cytokines in the respiratory airway as biomarkers of severity and prognosis for respiratory syncytial virus infection: an update. Front. Immunol. 2019;10:1154. doi: 10.3389/fimmu.2019.01154.
    1. Ugonna K, Douros K, Bingle CD, Everard ML. Cytokine responses in primary and secondary respiratory syncytial virus infections. Pediatr. Res. 2016;79:946–950. doi: 10.1038/pr.2016.29.
    1. Kutter JS, Spronken MI, Fraaij PL, Fouchier RA, Herfst S. Transmission routes of respiratory viruses among humans. Curr. Opin. Virol. 2018;28:142–151. doi: 10.1016/j.coviro.2018.01.001.
    1. Chatterjee A, Mavunda K, Krilov LR. Current state of respiratory syncytial virus disease and management. Infect. Dis. Ther. 2021;10:5–16. doi: 10.1007/s40121-020-00387-2.
    1. Staebler S, Blake S. Respiratory syncytial virus disease: Immunoprophylaxis policy review and public health concerns in preterm and young infants. Policy Polit. Nurs. Pract. 2021;22:41–50. doi: 10.1177/1527154420965543.
    1. Anderson LJ, et al. Strategic priorities for respiratory syncytial virus (RSV) vaccine development. Vaccine. 2013;31:B209–215. doi: 10.1016/j.vaccine.2012.11.106.
    1. Domachowske JB, Anderson EJ, Goldstein M. The future of respiratory syncytial virus disease prevention and treatment. Infect. Dis. Ther. 2021;10:47–60. doi: 10.1007/s40121-020-00383-6.
    1. Ruhe JJ, Hasbun R. Streptococcus pneumoniae bacteremia: Duration of previous antibiotic use and association with penicillin resistance. Clin. Infect. Dis. 2003;36:1132–1138. doi: 10.1086/374556.
    1. Behzadi MA, Leyva-Grado VH. Overview of current therapeutics and novel candidates against influenza, respiratory syncytial virus, and middle east respiratory syndrome Coronavirus infections. Front. Microbiol. 2019;10:1327. doi: 10.3389/fmicb.2019.01327.
    1. Taylor SL, et al. Long-term azithromycin reduces Haemophilus influenzae and increases antibiotic resistance in severe asthma. Am. J. Respir. Crit. Care Med. 2019;200:309–317. doi: 10.1164/rccm.201809-1739OC.
    1. Harun AM, et al. The antimicrobial properties of nanotitania extract and its role in inhibiting the growth of Klebsiella pneumonia and Haemophilus influenza. Antibiotics. 2021;10:961. doi: 10.3390/antibiotics10080961.
    1. Shahbazi R, Yasavoli-Sharahi H, Alsadi N, Ismail N, Matar C. Probiotics in treatment of viral respiratory infections and neuroinflammatory disorders. Molecules. 2020;25:4891. doi: 10.3390/molecules25214891.
    1. Lehtoranta L, Pitkäranta A, Korpela R. Probiotics in respiratory virus infections. Eur. J. Clin. Microbiol. Infect. Dis. 2014;33:1289–1302. doi: 10.1007/s10096-014-2086-y.
    1. Starosila D, Rybalko S, Varbanetz L, Ivanskaya N, Sorokulova I. Anti-influenza activity of a Bacillus subtilis probiotic strain. Antimicrob. Agents Chemother. 2017 doi: 10.1128/AAC.00539-17.
    1. Salminen S, et al. The international scientific association of probiotics and prebiotics (ISAPP) consensus statement on the definition and scope of postbiotics. Nat. Rev. Gastroenterol. Hepatol. 2021;18:649–667. doi: 10.1038/s41575-021-00440-6.
    1. Rautava S, Salminen S, Isolauri E. Specific probiotics in reducing the risk of acute infections in infancy-a randomised, double-blind, placebo-controlled study. Br. J. Nutr. 2009;101:1722–1726. doi: 10.1017/S0007114508116282.
    1. Harata G, et al. Intranasal administration of Lactobacillus rhamnosus GG protects mice from H1N1 influenza virus infection by regulating respiratory immune responses. Lett. Appl. Microbiol. 2010;50:597–602. doi: 10.1111/j.1472-765X.2010.02844.x.
    1. Hatakka K, et al. Effect of long term consumption of probiotic milk on infections in children attending day care centres: Double blind, randomised trial. BMJ. 2001;322:1327. doi: 10.1136/bmj.322.7298.1327.
    1. Kumpu M, et al. Milk containing probiotic Lactobacillus rhamnosus GG and respiratory illness in children: A randomized, double-blind, placebo-controlled trial. Eur. J. Clin. Nutr. 2012;66:1020–1023. doi: 10.1038/ejcn.2012.62.
    1. Luoto R, et al. Prebiotic and probiotic supplementation prevents rhinovirus infections in preterm infants: A randomized, placebo-controlled trial. J. Allergy Clin. Immunol. 2014;133:405–413. doi: 10.1016/j.jaci.2013.08.020.
    1. Liu S, Hu P, Du X, Zhou T, Pei X. Lactobacillus rhamnosus GG supplementation for preventing respiratory infections in children: A meta-analysis of randomized, placebo-controlled trials. Indian Pediatr. 2013;50:377–381. doi: 10.1007/s13312-013-0123-z.
    1. Anaya-Loyola MA, et al. Bacillus coagulans GBI-30, 6068 decreases upper respiratory and gastrointestinal tract symptoms in healthy Mexican scholar-aged children by modulating immune-related proteins. Food Res. Int. 2019;125:108567. doi: 10.1016/j.foodres.2019.108567.
    1. Kawase M, et al. Heat-killed Lactobacillus gasseri TMC0356 protects mice against influenza virus infection by stimulating gut and respiratory immune responses. FEMS Immunol. Med. Microbiol. 2012;64:280–288. doi: 10.1111/j.1574-695X.2011.00903.x.
    1. Garcia-Crespo KE, et al. Lactobacillus priming of the respiratory tract: Heterologous immunity and protection against lethal pneumovirus infection. Antivir. Res. 2013;97:270–279. doi: 10.1016/j.antiviral.2012.12.022.
    1. Setlow P. Spores of Bacillus subtilis: Their resistance to and killing by radiation, heat and chemicals. J. Appl. Microbiol. 2006;101:514–525. doi: 10.1111/j.1365-2672.2005.02736.x.
    1. Tan IS, Ramamurthi KS. Spore formation in Bacillus subtilis. Environ. Microbiol. Rep. 2014;6:212–225. doi: 10.1111/1758-2229.12130.
    1. Elshaghabee FMF, Rokana N, Gulhane RD, Sharma C, Panwar H. Bacillus as potential probiotics: Status, concerns, and future perspectives. Front. Microbiol. 2017;8:1490. doi: 10.3389/fmicb.2017.01490.
    1. Lee NK, Kim WS, Paik HD. Bacillus strains as human probiotics: Characterization, safety, microbiome, and probiotic carrier. Food Sci. Biotechnol. 2019;28:1297–1305. doi: 10.1007/s10068-019-00691-9.
    1. Slivnik M, et al. Effect of daily Bacillus subtilis DE111 intake on gastrointestinal health and respiratory infections in children attending day-care: A randomised, parallel, double-blind, placebo-controlled study. J. Probiotics Health. 2020;8:225.
    1. Song M, et al. Killed Bacillus subtilis spores as a mucosal adjuvant for an H5N1 vaccine. Vaccine. 2012;30:3266–3277. doi: 10.1016/j.vaccine.2012.03.016.
    1. Hong JE, et al. Alveolar macrophages treated with Bacillus subtilis spore protect mice infected with respiratory syncytial virus A2. Front. Microbiol. 2019;10:447. doi: 10.3389/fmicb.2019.00447.
    1. Wiegers HMG, et al. Bacterial co-infection of the respiratory tract in ventilated children with bronchiolitis; a retrospective cohort study. BMC Infect. Dis. 2019;19:938. doi: 10.1186/s12879-019-4468-3.
    1. Krishnan P, et al. Evaluation of coamoxiclav and other antibiotics against S. pneumoniae and H. influenae from paediatric cases of acute respiratory infections. J. Indian Med. Assoc. 2011;109:241–242.
    1. Jeannoel M, et al. Microorganisms associated with respiratory syncytial virus pneumonia in the adult population. Eur. J. Clin. Microbiol. Infect. Dis. 2019;38:157–160. doi: 10.1007/s10096-018-3407-3.
    1. Jennings LC, et al. Incidence and characteristics of viral community-acquired pneumonia in adults. Thorax. 2008;63:42–48. doi: 10.1136/thx.2006.075077.
    1. Edouard S, et al. The nasopharyngeal microbiota in patients with viral respiratory tract infections is enriched in bacterial pathogens. Eur. J. Clin. Microbiol. Infect. Dis. 2018;37:1725–1733. doi: 10.1007/s10096-018-3305-8.
    1. Garcia C, et al. Decreased innate immune cytokine responses correlate with disease severity in children with respiratory syncytial virus and human rhinovirus bronchiolitis. Pediatr. Infect. Dis. J. 2012;31:86–89. doi: 10.1097/INF.0b013e31822dc8c1.
    1. Cui AH, Zhao J, Liu SX, Hao YS. Associations of IL-4, IL-6, and IL-12 levels in peripheral blood with lung function, cellular immune function, and quality of life in children with moderate-to-severe asthma. Medicine. 2017;96:e6265. doi: 10.1097/MD.0000000000006265.
    1. Sheng YJ, et al. Low birth weight contributed to increased serum IL-6 levels in infantile respiratory syncytial virus infection. BMC Pediatr. 2017;17:205. doi: 10.1186/s12887-017-0961-2.
    1. Dienz O, Rincon M. The effects of IL-6 on CD4 T cell responses. Clin. Immunol. 2009;130:27–33. doi: 10.1016/j.clim.2008.08.018.
    1. McAllister CS, et al. Dexamethasone inhibits respiratory syncytial virus-driven mucus production while increasing viral replication without altering antiviral interferon signaling. Virology. 2020;540:195–206. doi: 10.1016/j.virol.2019.10.007.
    1. Sadeghi A, Mortazavi SA, Bahrami AR, Sadeghi B, Matin MM. Designing a SYBR Green absolute real time PCR assay for specific detection and quantification of Bacillus subtilis in dough used for bread making. JCMR. 2014;6:83–92.
    1. Perotti M, et al. Quantitation of Bacillus clausii in biological samples by real-time polymerase chain reaction. J. Microbiol. Methods. 2006;65:632–636. doi: 10.1016/j.mimet.2005.10.010.

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