Probiotic Lactobacillus Paracasei Expressing a Nucleic Acid-Hydrolyzing Minibody (3D8 Scfv) Enhances Probiotic Activities in Mice Intestine as Revealed by Metagenomic Analyses

The virus behind the current pandemic, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is responsible for the etiology of novel coronavirus disease (COVID-19) and poses a critical public health threat worldwide. Effective therapeutics and vaccines against multiple coronaviruses remain unavailable. Single-chain variable fragment (scFv), a recombinant antibody, exhibits broad-spectrum antiviral activity against DNA and RNA viruses owing to its nucleic acid-hydrolyzing property. The antiviral activity of 3D8 scFv against SARS-CoV-2 and other coronaviruses was evaluated in Vero E6 cell cultures. Viral growth was quantified with quantitative RT-qPCR and plaque assay. The nucleic acid-hydrolyzing activity of 3D8 was assessed through abzyme assays of in vitro viral transcripts and cell viability was determined by MTT assay. We found that 3D8 inhibited the replication of SARS-CoV-2, human coronavirus OC43 (HCoV-OC43), and porcine epidemic diarrhea virus (PEDV). Our results revealed the prophylactic and therapeutic effects of 3D8 scFv against SARS-CoV-2 in Vero E6 cells. Immunoblot and plaque assays showed the reduction of coronavirus nucleoproteins and infectious particles, respectively, in 3D8 scFv-treated cells. These data demonstrate the broad-spectrum antiviral activity of 3D8 against SARS-CoV-2 and other coronaviruses. Thus, it could be considered a potential antiviral countermeasure against SARS-CoV-2 and zoonotic coronaviruses.

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Avian influenza virus transmission is suppressed in chickens fed Lactobacillus paracasei expressing the 3D8 single-chain variable fragment protein

Acta Veterinaria Hungarica ◽

10.1556/004.2019.060 ◽

2019 ◽

Vol 67 (4) ◽

pp. 610-618

Author(s):

Hoonsung Choi ◽

Sang In Lee ◽

Shanmugam Sureshkumar ◽

Mi-Hyang Jeon ◽

Jeom Sun Kim ◽

...

Keyword(s):

Influenza Virus ◽

Avian Influenza ◽

Avian Influenza Virus ◽

Nuclease Activity ◽

Lactobacillus Paracasei ◽

Post Inoculation ◽

Single Chain Variable Fragment ◽

Single Chain ◽

Contact Transmission ◽

3D8 Scfv

The 3D8 single-chain variable fragment (scFv) is a mini-antibody sequence with independent nuclease activity that shows antiviral effects against all types of viruses in chickens and mice. In this study, chickens were treated daily with an oral dose of 109 CFU Lactobacillus paracasei (L. paracasei) expressing either a secreted or anchored 3D8 scFv for three weeks. After L. paracasei administration, the chickens were challenged with avian influenza virus (AIV). From each experimental group, three chickens were directly infected with 100 µL of 107.5 EID50/mL H9N2 AIV and seven chickens were indirectly challenged through contact transmission. oropharyngeal and cloacal swab samples were collected at 3, 5, 7, and 9 days post-inoculation (dpi) from AIV-challenged chickens, AIV Shedding titres were measured by quantitative real-time PCR. Contact transmission in the chickens that were fed 3D8 scFv-secreting L. paracasei showed a significant reduction in viral shedding when compared with other groups. These results suggest that L. paracasei secreting 3D8 provides a basis for the development of ingestible antiviral probiotics with activity against AIV.

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To the editor: probiotic Lactobacillus paracasei effect on cariogenic bacterial flora

Clinical Oral Investigations ◽

10.1007/s00784-011-0660-6 ◽

2011 ◽

Vol 16 (5) ◽

pp. 1513-1513

Author(s):

Alireza Akbarzadeh Baghban ◽

Leili Mehrdad

Keyword(s):

Bacterial Flora ◽

Lactobacillus Paracasei ◽

Probiotic Lactobacillus

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Probiotic Lactobacillus paracasei effect on cariogenic bacterial flora

Clinical Oral Investigations ◽

10.1007/s00784-010-0423-9 ◽

2010 ◽

Vol 15 (4) ◽

pp. 471-476 ◽

Cited By ~ 43

Author(s):

Li-Chuan Chuang ◽

Chiung-Shing Huang ◽

Li-Wei Ou-Yang ◽

Shiao-Yu Lin

Keyword(s):

Bacterial Flora ◽

Lactobacillus Paracasei ◽

Probiotic Lactobacillus

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Effect of Fluidized Bed Drying, Matrix Constituents and Structure on the Viability of Probiotic Lactobacillus paracasei ATCC 55544 during Storage at 4 °C, 25 °C, and 37 °C

Microorganisms ◽

10.3390/microorganisms10010074 ◽

2021 ◽

Vol 10 (1) ◽

pp. 74

Author(s):

Devastotra Poddar ◽

Jon Palmer ◽

Shantanu Das ◽

Manju Gaare ◽

Arup Nag ◽

...

Keyword(s):

Fluidized Bed ◽

Milk Powder ◽

Laser Scanning Microscopy ◽

Lactobacillus Paracasei ◽

Confocal Laser ◽

Whole Milk ◽

Probiotic Lactobacillus ◽

Fluidized Bed Drying ◽

The Impact ◽

Whole Milk Powder

The stabilization of probiotics for application in non-refrigerated food products is a challenging task. In the present study, bacteria were immobilized in a dairy matrix comprising of whole milk powder, skim milk powder, or milk protein isolate using fluidized bed drying technology. The samples were taken out at different drying stages, with an apparent water activity of aw 0.5, aw 0.4, and aw 0.3, respectively, and vacuum-packed to maintain the aw and stored at three different temperatures of 4 °C, 25 °C, and 37 °C. The study evaluated the impact of matrix constituents, milk fat, protein, and carbohydrate on the viability of encapsulated probiotic Lactobacillus paracasei (Lacticaseibacillus paracasei) ATCC 55544 during storage for 1 month. The whole milk powder matrix provided superior protection to the bacteria. Confocal Laser Scanning Microscopy (CLSM) was used to investigate the structure of the immobilizing matrix and the location of the probiotic L. paracasei cells embedded within the matrix. The CLSM study revealed that the probiotic bacterial cells are mostly embedded as clusters beneath the top layer. We hypothesize that the biofilm-like structure, together with the protective whole milk powder matrix, helps to retain the superior viability of probiotic cells during storage at non-refrigerated storage conditions of 25 °C and 37 °C.

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3D8, a nucleic acid-hydrolyzing scFv, confers antiviral activity against SARS-CoV-2 and multiple coronaviruses in vitro

10.1101/2020.11.25.398909 ◽

2020 ◽

Author(s):

Gunsup Lee ◽

Shailesh Budhathoki ◽

Hyeok Soon Choi ◽

Kwang-ji Oh ◽

Geum-Young Lee ◽

...

Keyword(s):

Nucleic Acid ◽

Antiviral Activity ◽

Broad Spectrum ◽

Rna Viruses ◽

Therapeutic Effects ◽

List Type ◽

Single Chain ◽

Diarrhea Virus ◽

Antiviral Effects ◽

3D8 Scfv

AbstractThe current pandemic severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pose a critical public health threat worldwide. Coronaviruses (subfamily Orthocoronavirinae, family Coronaviridae, order Nidovirales) are a group of enveloped positive-sense single-stranded RNA viruses. Six pathogenic human coronaviruses, likely zoonotic viruses, cause the common cold in humans. A new emerging coronavirus, SARS-CoV-2, become a crucial etiology for the Coronavirus-induced disease 19 (COVID-19). However, effective therapeutics and vaccines against multiple coronaviruses remain unavailable. This study aimed to investigate an antiviral molecule, single chain variable fragment (scFv), against SARS-CoV-2 and other coronaviruses. 3D8, a recombinant scFv, exhibits broad-spectrum antiviral activity against DNA and RNA viruses owing to its nucleic acid-hydrolyzing property. Here, we report that 3D8 scFv inhibited the replication of SARS-CoV-2, human coronavirus OC43 (HCoV-OC43), and porcine epidemic diarrhea virus (PEDV). Our results revealed the prophylactic and therapeutic effects of 3D8 scFv against SARS-CoV-2 in Vero E6 cells. Immunoblot and plaque assays showed the absence of coronavirus nucleoproteins and infectious particles in 3D8 scFv-treated cells, respectively. In addition, we observed the antiviral effects of 3D8 against HCoV-OC43 and PEDV. In conclusion, this study provides insights into the broad-spectrum antiviral agent of 3D8 scFv; thus, it could be considered a potential antiviral countermeasure against SARS-CoV-2 and zoonotic coronaviruses.Key points (Main message)3D8, a nucleic acid-hydrolyzing scFv, exhibits potent prophylactic and therapeutic antiviral effects on SARS-CoV-2.3D8 exhibits broad-spectrum antiviral activity against multiple coronaviruses: hCoV OC43 and PEDV.3D8 potentially degrades viral RNA.

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