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Stable expression of HIV-1 MPER extended epitope on the surface of the recombinant probiotic bacteria Escherichia Coli Nissle 1917 using CRISPR/Cas9
Örebro University, School of Medical Sciences.ORCID iD: 0000-0001-6819-7960
School of Science and Technology, Life Science Center, Örebro University, Örebro, Sweden; Institute of Virology, Saarland University Medical Center, 66421, Homburg, Germany.
Örebro University, School of Science and Technology. (Life Science Center)
Division of Clinical Microbiology, Department of Laboratory Medicine, Karolinska Institutet, Stockholm, Sweden; Department of Microbiology, Public Health Agency of Sweden, Solna, Sweden.
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2024 (English)In: Microbial Cell Factories, E-ISSN 1475-2859, Vol. 23, no 1, article id 39Article in journal (Refereed) Published
Abstract [en]

BACKGROUND: Mucosal vaccines have the potential to induce protective immune responses at the sites of infection. Applying CRISPR/Cas9 editing, we aimed to develop a probiotic-based vaccine candidate expressing the HIV-1 envelope membrane-proximal external region (MPER) on the surface of E. coli Nissle 1917.

RESULTS: The HIV-1 MPER epitope was successfully introduced in the porin OmpF of the E. coli Nissle 1917 (EcN-MPER) and the modification was stable over 30 passages of the recombinant bacteria on the DNA and protein level. Furthermore, the introduced epitope was recognized by a human anti-HIV-1 gp41 (2F5) antibody using both live and heat-killed EcN-MPER, and this antigenicity was also retained over 30 passages. Whole-cell dot blot suggested a stronger binding of anti-HIV-1 gp41 (2F5) to heat-killed EcN-MPER than their live counterpart. An outer membrane vesicle (OMV) - rich extract from EcN-MPER culture supernatant was equally antigenic to anti-HIV-1 gp41 antibody which suggests that the MPER antigen could be harboured in EcN-MPER OMVs. Using quantitative ELISA, we determined the amount of MPER produced by the modified EcN to be 14.3 µg/108 cfu.

CONCLUSIONS: The CRISPR/Cas9 technology was an effective method for establishment of recombinant EcN-MPER bacteria that was stable over many passages. The developed EcN-MPER clone was devoid of extraneous plasmids and antibiotic resistance genes which eliminates the risk of plasmid transfer to animal hosts, should this clone be used as a vaccine. Also, the EcN-MPER clone was recognised by anti-HIV-1 gp41 (2F5) both as live and heat-killed bacteria making it suitable for pre-clinical evaluation. Expression of OmpF on bacterial surfaces and released OMVs identifies it as a compelling candidate for recombinant epitope modification, enabling surface epitope presentation on both bacteria and OMVs. By applying the methods described in this study, we present a potential platform for cost-effective and rational vaccine antigen expression and administration, offering promising prospects for further research in the field of vaccine development.

Place, publisher, year, edition, pages
BioMed Central (BMC), 2024. Vol. 23, no 1, article id 39
Keywords [en]
CRISPR/Cas9, HIV-1, Membrane-proximal external region (MPER), Outer membrane protein F (OmpF), Probiotic
National Category
Microbiology
Identifiers
URN: urn:nbn:se:oru:diva-111380DOI: 10.1186/s12934-023-02290-0ISI: 001157372700001PubMedID: 38311724Scopus ID: 2-s2.0-85184084053OAI: oai:DiVA.org:oru-111380DiVA, id: diva2:1834578
Funder
Knowledge Foundation, 20200063Örebro University
Note

Correction: Stable expression of HIV-1 MPER extended epitope on the surface of the recombinant probiotic bacteria Escherichia Coli Nissle 1917 using CRISPR/Cas9. Ninyio, N., Schmitt, K., Sergon, G. et al. Microb Cell Fact 23, 75 (2024). https://doi.org/10.1186/s12934-024-02347-8

Available from: 2024-02-05 Created: 2024-02-05 Last updated: 2024-03-08Bibliographically approved

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Ninyio, NathanielSergon, GladysAndersson, SörenScherbak, Nikolai

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