Stable expression of HIV-1 MPER extended epitope on the surface of the recombinant probiotic bacteria Escherichia Coli Nissle 1917 using CRISPR/Cas9

• Published in: Microbial cell factories 2024-02, Vol.23 (1), p.39-39, Article 39
• Main Authors: Ninyio, Nathaniel, Schmitt, Katherina, Sergon, Gladys, Nilsson, Charlotta, Andersson, Sören, Scherbak, Nikolai
• Format: Article
• Language: English
• Subjects: Analysis; Animal genetic engineering; Animals; Antibiotic resistance; Antibodies; Antigen presentation; Antigenic determinants; Antigenicity; Antigens; Antiviral agents; Bacteria; Cell culture; Cloning; Coliforms; CRISPR; CRISPR-Cas Systems; CRISPR/Cas9; Drug resistance in microorganisms; E coli; Enzyme-linked immunosorbent assay; Epitopes; Escherichia coli; Escherichia coli - genetics; Genetic aspects; Genomes; Glycoprotein gp41; Health aspects; Heat; HIV; HIV (Viruses); HIV Antibodies; HIV-1; HIV-1 - genetics; Human immunodeficiency virus; Humans; Identification and classification; Immune response; Immune system; Medical research; Medicin och hälsovetenskap; Medicine, Experimental; Membrane-proximal external region (MPER); Membranes; Methods; Molecular Biology; Molekylärbiologi; Monoclonal antibodies; Outer membrane protein F (OmpF); Plasmids; Porins; Probiotic; Probiotics; Proteins; Vaccine development; Vaccines; Viral antibodies
• ISSN: 1475-2859; 1475-2859
• DOI: 10.1186/s12934-023-02290-0

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. 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/10 cfu. 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.