The “humanized” N‐glycosylation pathway in CRISPR/Cas9‐edited Nicotiana benthamiana significantly enhances the immunogenicity of a S/preS1 Hepatitis B Virus antigen and the virus‐neutralizing antibody response in vaccinated mice

The recent SARS‐CoV‐2 pandemic has taught the world a costly lesson about the devastating consequences of viral disease outbreaks but also, the remarkable impact of vaccination in limiting life and economic losses. Vaccination against human Hepatitis B Virus (HBV), a major human pathogen affecting 290 million people worldwide, remains a key action towards viral hepatitis elimination by 2030. To meet this goal, the development of improved HBV antigens is critical to overcome non‐responsiveness to standard vaccines based on the yeast‐produced, small (S) envelope protein. We have recently shown that combining relevant immunogenic determinants of S and large (L) HBV proteins in chimeric antigens markedly enhances the anti‐HBV immune response. However, the demand for cost‐efficient, high‐quality antigens remains challenging. This issue could be addressed by using plants as versatile and rapidly scalable protein production platforms. Moreover, the recent generation of plants lacking β‐1,2‐xylosyltransferase and α‐1,3‐fucosyltransferase activities (FX‐KO), by CRISPR/Cas9 genome editing, enables production of proteins with “humanized” N‐glycosylation. In this study, we investigated the impact of plant N‐glycosylation on the immunogenic properties of a chimeric HBV S/L vaccine candidate produced in wild‐type and FX‐KO Nicotiana benthamiana. Prevention of β‐1,2‐xylose and α‐1,3‐fucose attachment to the HBV antigen significantly increased the immune response in mice, as compared with the wild‐type plant‐produced counterpart. Notably, the antibodies triggered by the FX‐KO‐made antigen neutralized more efficiently both wild‐type HBV and a clinically relevant vaccine escape mutant. Our study validates in premiere the glyco‐engineered Nicotiana benthamiana as a substantially improved host for plant production of glycoprotein vaccines.