Developing a Live Probiotic Vaccine Based on the Enterococcus faecium L3 Strain Expressing Influenza Neuraminidase
Abstract
:1. Introduction
2. Materials and Methods
2.1. Molecular Analysis
2.2. Obtaining a Chimeric Protein from E. faecium L3 d2 Gene and a Fragment of the NA Gene
2.3. Real-Time Reverse Transcriptase PCR (rRT-PCR)
2.4. Study of Expression of NA Protein on the Surface of E. faecium L3 Using ELISA
2.5. Immunoelectron Microscopy to Study the Structure of E. faecium L3 Pili with Expression of Viral Proteins
2.6. Oral Immunization of Mice with Live Probiotic Vaccine
2.7. Evaluation of L3-NA Persistence in the Gastrointestinal Tract of Vaccinated Mice
2.8. Ethic Statement
2.9. Determination of NA-Specific IgG and IgA
2.10. Study of Protection against Influenza Infection after Vaccination with Live Probiotic Vaccine
2.11. Statistics
3. Results
3.1. Molecular Analysis of NA Insertion
3.2. Generation of the Live Probiotic Influenza Vaccine Candidate
3.3. Expression Levels of the NA mRNA in the Bacteria
3.4. Immunoelectron Microscopy
3.5. Bacterial Persistence in the Intestines of CBA Mice
3.6. Immunogenicity in CBA Mice
3.7. Protection against Lethal Influenza Infection
3.8. Immunogenicity and Protection against Virus-Bacterial Infection in Balb/c Mice
4. Discussion
5. Conclusions
Supplementary Materials
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Acknowledgments
Conflicts of Interest
References
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Subtype/Lineage | Name | Sequence Number |
---|---|---|
H2N2 | A/Leningrad/134/17/1957 | L37330 |
H2N2 | A/duck/Pennsylvania/14-030488-005/2014 | QHQ82622 |
H3N2 | A/17/Singapore/2016/3571 | EPI1313135 |
H3N2 | A/South Dakota/12/2020 | MT467157 |
H1N1 | A/California/07/2009 | HM138502 |
H1N1 | A/South Africa/A201-004-041/2018 | MN716456 |
H5N1 | A/Viet Nam/1203/2004 | ABP52008 |
H7N3 | A/chicken/Karachi/NARC-100/2004 | FJ577544 |
H7N9 | A/Anhui/1-DEWH730/2013 | CY187620 |
H7N9 | A/Guangdong/HP001/2017 | AQY18948 |
B/Victoria | B/Brisbane/60/2008 | AFH57913 |
B/Yamagata | B/Wisconsin/04/2015 | ALH38476 |
Primers | Direction | Nucleotide Sequence from 5′ to 3′ |
---|---|---|
EV | forward | GCATATGGCGATCCTGGCAAGTGTTAT 1 |
FV | reverse | GGAATTCCTAGAGCTGTCGTCGTTCC |
A1 | forward | GCTCTAGAGCCGATGAGAGCAGCTGGTATTG |
D1 | reverse | CAACAGGATCCAAAGCATCGTTGG |
B1 | forward | TGAGTGAACCACAGCCAGAA |
Seq F | forward | GGACACCACAACCATCGAAG |
Seq R | reverse | AGCTGGACCATGCTACACA |
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Desheva, Y.; Leontieva, G.; Kramskaya, T.; Gupalova, T.; Losev, I.; Kuleshevich, E.; Bormotova, E.; Kopteva, O.; Kudar, P.; Suvorov, A. Developing a Live Probiotic Vaccine Based on the Enterococcus faecium L3 Strain Expressing Influenza Neuraminidase. Microorganisms 2021, 9, 2446. https://doi.org/10.3390/microorganisms9122446
Desheva Y, Leontieva G, Kramskaya T, Gupalova T, Losev I, Kuleshevich E, Bormotova E, Kopteva O, Kudar P, Suvorov A. Developing a Live Probiotic Vaccine Based on the Enterococcus faecium L3 Strain Expressing Influenza Neuraminidase. Microorganisms. 2021; 9(12):2446. https://doi.org/10.3390/microorganisms9122446
Chicago/Turabian StyleDesheva, Yulia, Galina Leontieva, Tatiana Kramskaya, Tatiana Gupalova, Igor Losev, Eugenia Kuleshevich, Elena Bormotova, Olga Kopteva, Polina Kudar, and Alexander Suvorov. 2021. "Developing a Live Probiotic Vaccine Based on the Enterococcus faecium L3 Strain Expressing Influenza Neuraminidase" Microorganisms 9, no. 12: 2446. https://doi.org/10.3390/microorganisms9122446