Ana‐Maria Pantazica,André van Eerde,Mihaela‐Olivia Dobrica,Iuliana Caras,Irina Ionescu,Adriana Costache,Catalin Tucureanu,Hege Steen,Catalin Lazar,Inger Heldal,Sissel Haugslien,Adrian Onu,Crina Stavaru,Norica Branza‐Nichita,Jihong Liu Clarke
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 Ana‐Maria Pantazica et al.
Nicotiana benthamiana,CRISPR/Cas9,HBV vaccine,plant N‐glycosylation,FX‐KO,HBV
Summary 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.
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 Ana‐Maria Pantazica et al.

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.

Despite major advances in the current understanding of Hepatitis B Virus (HBV) infection and pathology, a curative treatment is still missing and more than 800 000 chronically infected patients are losing the battle with this disease every year (WHO, 2022). Vaccination against HBV remains a key action of the World Health Organization's (WHO) global strategy towards elimination of viral hepatitis as a public health concern, by 2030 (WHO, 2016). Currently marketed HBV vaccines, based on expression of the small (S) envelope protein in yeast and assembly of subviral particles (SVPs), also known as the S‐HBsAg, trigger a protective humoral immune response in more than 90% of vaccinated adults (Rubin et al., 2014). However, the development of more immunogenic HBV antigens is critical to increase the immune response in poor‐ or non‐responders to the standard S‐based vaccine, who are at high risk for infection. In addition, vaccine responders may remain susceptible to infection with HBV variants containing mutations within the major antigenic determinant of the S protein, collectively denoted as vaccine‐escape mutations (VEMs), that are not neutralized by antibodies against the wild‐type (WT) S protein (Bian et al., 2013; Lai et al., 2012). To address these unsolved challenges of HBV vaccination, the FDA has recently approved a novel HBV vaccine incorporating all three envelope proteins, S, medium (M) and large (L), produced in mammalian cells (Vesikari et al., 2021a,b). Booster vaccination with this highly immunogenic formulation resulted in production of protective levels of anti‐HBsAg antibodies in previous non‐responders to the S only vaccine (Körber et al., 2021). The L envelope protein is a particularly attractive immunogen due to the presence of the N‐terminal preS1 domain, which is responsible for HBV attachment to its hepatocyte receptor, the sodium taurocholate co‐transporting polypeptide (NTCP) (Yan et al., 2012) and a target for virus‐neutralizing antibodies (Hong et al., 2004; Yato et al., 2021). The ability of the L‐HBsAg to also trigger an efficient immune response against clinically important VEMs has very recently been confirmed in rhesus macaques trials (Washizaki et al., 2022). As an alternative strategy to incorporating individual HBV envelope proteins in antigenic cocktails, we have developed chimeric S/L antigens by inserting preS1 sequences into the major antigenic domain of the S protein, therefore ensuring an equimolar display of relevant S‐ and L‐derived epitopes on the same molecule (Dobrica et al., 2017, 2018). Of the series of chimeric proteins produced in mammalian cells, the novel S/preS116‐42 antigen has shown enhanced immunogenicity when compared with WT S and a different pattern of humoral and cellular immune activation, resulting in production of antibodies with neutralizing activity against both WT and VEMs HBV variants (Pantazica et al., 2022). However, upscaling the production of HBV antigens in mammalian cells is expensive and technically cumbersome (Dobrica et al., 2021a); therefore, in this work, we investigated the suitability of Nicotiana benthamiana as an alternative, cost‐efficient host for expression of the newly developed S/preS116‐42 HBV vaccine candidate. By using our established vacuum infiltration‐based transient expression system, we have previously demonstrated that complex viral envelope glycoprotein folding, processing and assembly occur in N. benthamiana, as in mammalian cells (Dobrica et al., 2017, 2018). This technology is speedy, versatile and safe, as recombinant proteins are expressed exclusively in infiltrated leaves, while reproductive tissues are not affected, thus preventing potential transgene escape (Clarke et al., 2017). The great potential of this protein expression platform is illustrated by the recent release of a plant‐produced COVID‐19 vaccine, approved in Canada for adults age 18–64, representing the world's first plant‐based vaccine authorized for human use (National Advisory Committee on Immunization, 2022). N‐glycosylation is one of the most conserved post‐translational modification pathways in eukaryotic cells, with crucial roles in protein properties and functions (Reuter and Gabius, 1999). Although the early, endoplasmic reticulum (ER)‐specific, N‐glycan maturation steps are similar in plants and mammalian cells, notable processing differences between the two systems occur later on the secretory pathway, in the Golgi compartment. Unlike mammalian cells, plants synthesize complex N‐glycans with β‐1,2‐linked xylose (core xylose) and core α‐1,3‐linked fucose, while lacking sialic acid and core α‐1,6‐linked fucose (Lerouge et al., 1998; Strasser, 2016). Interestingly, it has been shown that plant‐specific N‐glycans are immunogenic in mammals, which raises concerns that prolonged use of plant‐made therapeutic glycoproteins may pose health risks in humans (Bardor et al., 2003; Faye et al., 1993). With the advance of powerful molecular‐ and genome‐editing technologies it has become possible to avoid addition of β‐1,2‐ xylose and core α‐1,3‐fucose epitopes on plant‐expressed glycoproteins, by knocking‐out corresponding transferases of the endogenous N‐glycosylation pathway (Hanania et al., 2017; Li et al., 2016; Mercx et al., 2017). Very recently, a multiplex CRISPR/Cas9 genome‐editing approach was successfully used to generate N. benthamiana lines lacking β‐1,2‐xylosyltransferase and α‐1,3‐fucosyltransferase activity (FX‐KO) (Jansing et al., 2019). Production of human monoclonal antibodies in the FX‐KO line further demonstrated the absence of plant‐specific N‐glycosylation and similar biological activity to the mammalian cell‐made counterparts (Frigerio et al., 2022; Jansing et al., 2019). However, to the best of our knowledge, the impact of this remarkable change of N‐glycan pattern on the properties of recombinant proteins that are designed to be antigenic (i.e., vaccine candidates) has not yet been investigated in these plants. HBV‐S and the chimeric proteins derived from it are ideal model antigens to investigate this potential influence, due to the presence of a single N‐glycan within the antigenic domain and the well‐documented role of N‐glycosylation in modulating the immune response and evasion (Dobrica et al., 2020; Hyakumura et al., 2015; Yu et al., 2014). Therefore, in our present study, we took advantage of the availability of the FX‐KO N. benthamiana lines to produce the S/preS116‐42 HBV antigen and perform a comprehensive, side‐by‐side comparison of the molecular, antigenic and immunogenic properties of the protein obtained in the glycosylation mutant versus the wild type (WT). Analysis of the N‐glycan profile by mass spectrometry demonstrated the plant‐specific complex glycosylation of the WT N. benthamiana‐derived antigen while β‐1,2‐ xylose and core α‐1,3‐fucose attachment was prevented in the FX‐KO line, as expected. Absence of β‐1,2‐ xylose and α‐1,3‐fucose from the HBV antigen significantly increased the immune response in vaccinated mice, as compared with the WT plant‐expressed counterpart. Importantly, the antibodies triggered by the FX‐KO antigen had superior virus neutralizing capacity against both WT HBV and a clinically relevant VEM. Our study validates in premiere the glyco‐engineered N. benthamiana as a substantially improved host for plant production of glycoprotein vaccine candidates.

The possibility to produce the novel HBV antigen in N. benthamiana was determined by using the standard WT and a newly generated CRISPR/Cas9‐edited plant line with genetic knockouts of two 1,2‐xylosyltransferase and four α‐1,3‐fucosyltransferase genes, respectively (Jansing et al., 2019). Expression of the HBV S/preS116‐42 antigen in plants was first tested in WT N. benthamiana by using vacuum‐based agroinfiltration with the corresponding expression plasmid. Proteins were extracted from leaves harvested at day 6 post‐infiltration and subjected to western blot analysis using anti‐preS1 antibodies. Both N‐glycosylated (gp) and non‐glycosylated (p) forms of the HBV antigen were readily detectable in plant extracts, similar to mammalian cells (Figure 1a, left panel). Detection of known amounts of the HBV S/preS116‐42 antigen purified from supernatants of transfected HEK 293 T cells is also shown for comparison (Figure 1a, right panel) (Pantazica et al., 2022). Co‐expression of these glycoforms is a distinct feature of the S protein, owing to the presence of a single N‐glycosylation site at residue N146 that is occupied on a fraction of the envelope proteins in physiological HBV infection or recombinant systems (Julithe et al., 2014). This demonstrates that N. benthamiana is compatible with expression of the novel chimeric HBV antigen, justifying the investigation of the FX‐KO line as a production host for comparative studies. Similar to WT plants, the FX‐KO line was able to produce the HBV S/preS116‐42 antigen with no apparent differences in appearance of the agroinfiltrated leaves (Figure 1b, left panel). The HBV antigen accumulation in the FX‐KO line occurred at a noticeably lower rate than in the WT plants (Figure 1b, middle panel). Based on this observation, large‐scale agroinfiltration experiments were performed for protein purification where the leaves were harvested at 6 or 7 days post‐infiltration (dpi) for WT N. benthamiana and 8 days for the FX‐KO line (Figure 1b, right panel). The average expression level of the HBV antigen in these leaf extracts was ~200 μg/g fresh weight. This is significantly higher than previously reported S‐HBsAg levels achieved by Agrobacterium infiltration in various plant hosts (Pniewski, 2013) and close to those obtained by using the powerful expression system based on plant viral vectors (Huang et al., 2008). In mammalian cells, the HBV S/preS116‐42 antigen undergoes complex folding in the ER and assembly in SVPs that are exported from cells via the constitutive secretory pathway (Pantazica et al., 2022). To determine whether manipulation of the N‐glycosylation pathway in the FX‐KO cells had any impact on the plant folding machinery, expression of BiP, a highly conserved molecular chaperone and the key regulator of ER stress signalling in mammalian and plant cells, was further analysed in N. benthamiana leaf extracts. Detection of the housekeeping protein, GAPDH, was used as a protein loading control. As shown in Figure 1c, BiP expression increased in both WT and FX‐KO samples producing the HBV antigen, as compared with plants transiently transformed by using vacuum‐based agroinfiltration with the empty expression vector, likely as a consequence of recombinant protein accumulation and retention within cells (Leborgne‐Castel et al., 1999). In contrast, GAPDH expression was fairly similar in all samples supporting the specificity of BiP overexpression in plants producing the HBV antigen. Notably, BiP activation occurred at similar levels in the two plant lines, indicating that the altered genetic background of the FX‐KO plants did not affect their capacity to respond to ER stress and manage the synthesis of large amounts of foreign proteins. This concept was further validated following analysis of cell lysates by SDS‐PAGE under non‐reducing (‐DTT) conditions and a sucrose‐gradient ultracentrifugation, respectively, indicating a similar oligomerization pattern (Figure 2a) and assembly of HBV S/preS116‐42 antigen in SVPs (Figure 2b) in WT and FX‐KO plants.

Plant extracts were first subjected to 20% sucrose‐bed ultracentrifugation to concentrate S/preS116‐42‐SVPs, followed by fractionation on a 15%–60% sucrose gradient and gel filtration of antigen‐positive fractions on CaptoCore 400. Samples were concentrated and analysed by SDS‐PAGE under reducing conditions, followed by either Coomassie staining (Figure 3a) or western blot and detection with anti‐preS1 antibodies (Figure 3b). These analyses revealed the presence of a major, broad band at ~25 kDa, the expected molecular mass of the monomeric antigen and of weaker, higher molecular weight bands, likely corresponding to protein dimers and oligomers, reflecting the complexity of S/preS116‐42 folding. Total protein and S/preS116‐42 quantification after the purification steps indicated an antigen purity of ~80%, confirming the Coomassie staining (Figure 3a). The antigen monomers from WT and FX‐KO N. benthamiana‐derived samples were further extracted from gels and treated with PNGase A to release the N‐glycans, followed by UPLC‐FLD/ESI‐TOF MS analysis of free glycan structures. Fluorescence detection permitted detection of uniformly labelled glycan peaks with very high sensitivity, while mass detection provided structural data for the assignment of peaks to glycan structures. For each peak, the mass was determined by the ESI‐MS spectrum at the corresponding retention time. Also, a mass distribution spectrum (single ion chromatogram) was assigned to the glycan, confirming the mass found against the fluorescence chromatogram of the sample containing all peaks. For quantification, peak areas were integrated and converted into relative percentages of the total glycan amount, defined as the sum of all peaks and normalized to 100%. A substantially different N‐glycan pattern was observed between the two antigen samples, as summarized in Tables 1 and 2. The mass spectrum of WT‐derived S/preS116‐42 displayed a major peak (~63% of the total glycans), representing the plant‐specific complex N‐glycan N3H4FX, containing β‐1,2‐xylose and core α‐1,3‐fucose (Figure 3c, Table 1). The remaining peaks contained mostly complex glycan without xylose and fucose, while only ~11% of the glycans were partially processed, high‐mannose N2H5 structures. In contrast, the N4H5 lacking β‐1,2‐xylose and core α‐1,3‐fucose was the predominant complex N‐glycan attached to the HBV antigen produced in FX‐KO plants (~55%, Figure 3d, Table 2). Other mammalian cell‐specific complex N‐glycans, such as N4H4 and N3H3 were also more abundant in this sample (Figure 3d, Table 2). Apparently, xylosylated and core α‐1,3‐fucosylated structures were still produced in these plants, however, at very low abundance (0.7–1.4% of the total N‐glycans). These data describe the first detailed N‐glycan analysis of an HBV antigen produced in plant cells and indicate that S/preS116‐42 particles are transported along the secretory pathway to the trans‐Golgi network where the N‐linked glycans are processed to complex structures by specific glycosyltransferases.

To determine the potential impact of host‐specific N‐glycosylation on the antigenicity of the HBV S/preS116‐42 protein, binding of the protein to a panel of commercially available, conformation‐dependent anti‐S antibodies was analysed by ELISA. These monoclonal antibodies were raised in mice following immunization with purified HBV or S‐HBsAg. As expected, the most efficient binding was observed for the antigen obtained in HEK293T cells, which likely recapitulates with higher fidelity the immunogenic determinants of the injected antigen. Notably, the S/preS116‐42 antigen produced in WT N. benthamiana displayed lower binding regardless of the antibody used (Figure 4). A significant improvement of antibody reactivity was observed for the antigen expressed in FX‐KO plants, approaching that obtained for the protein produced in mammalian cells, as in the case of the #10–1323 antibodies (Figure 4b). Although the exact identity of the viral epitopes recognized by this panel of antibodies is not known, the host‐dependent difference in antigen‐binding activity suggests that the N‐glycan structure plays an important role in this process. However, as the N‐glycan partial “humanization” in FX‐KO plants did not fully recover the mammalian cell‐specific antigenicity of S/preS116‐42, a slightly different conformation of the antigen between the three expression systems cannot be completely ruled out.

The immunogenicity of WT or FX‐KO N. benthamiana‐expressed HBV‐S/preS116‐42 was evaluated in mice following intramuscular administration of 25 μg of antigen in the presence of AddaVax, a squalene‐based oil‐in‐water emulsion. This class of adjuvants has been shown to protect the antigen from premature clearing and activate both cellular and humoral immune responses, resulting in improved immune protection in humans (O'Hagan et al., 2021). Mice sera were collected before prime (day 0) and after the first (day 28) and second boost (day 42), respectively, then tested for the presence of HBV‐specific IgG and IgG subclasses. As shown in Figure 6a, both HBV antigens elicited a significant amount of IgG antibodies already after the first boost, with higher titers observed in mice immunized with the protein purified from FX‐KO N. benthamiana. This trend was maintained at day 42, when the immune response elicited by the FX‐KO N. benthamiana‐derived S/preS116‐42 was up to 4‐folds higher as compared with that induced by the antigen produced in WT plants (Figure 5a). Analysis of IgG1 and IgG2a as markers of humoral and cellular immune activation, respectively, confirmed the superior immunogenicity of the FX‐KO N. benthamiana‐ produced HBV antigen, which triggered significantly higher levels of either antibody subclass as compared with the WT‐derived counterpart, at all checkpoints (Figure 5, panels a and b). Notably, a more prominent increase was observed for IgG1 titers, suggesting a main polarization of the Th2‐T cell type immune response, consistent with the properties of the HBsAg. To further investigate the cellular immune response induced by the two antigens, spleen cells from control and immunized mice were stimulated with phosphate buffered saline (PBS) (negative control, N) or HBV particles (Ag). Concanavalin A (CONA) and CD3/CD28 antibodies (CD3/CD28) were also included as positive controls of cell activation. Secretion of IFN‐γ and IL‐5, signature cytokines of Th1‐ and Th2‐T cell type activation, respectively, was determined by ELISpot. HBV stimulation significantly increased the number of IFN‐γ‐secreting spleen cells from mice that received the chimeric antigen, as compared with the control group that received the adjuvant only (Figure 6a, left panel). A similar behaviour was noted for IL‐5‐secreting splenocytes (Figure 6b, left panel). In both cases, the strongest immune response was observed in mice immunized with FX‐KO N. benthamiana‐S/preS116‐42. In contrast, IFN‐γ and IL‐5 secretion from PBS‐stimulated spleen cells derived from mice immunized with either antigen was virtually undetectable, demonstrating the specificity and reactivity of the cellular immune response towards HBV (Figure 6).

The quality of a vaccine is ultimately demonstrated by the efficacy of the triggered immune response in preventing infection. Therefore, the capacity of antibodies raised by immunization with WT and FX‐KO N. benthamiana‐derived S/preS116‐42 to protect from HBV infection was next investigated in vitro. WT HBV and a VEM variant containing the G145R mutation within the S domain, which is most frequently identified in clinical samples (Zuckerman, 2000), were produced in Huh7 cells as described previously (Pantazica et al., 2022). WT and VEM virus inocula were treated with pre‐ or post‐immune sera (1:50 dilution) collected from mice vaccinated with either antigen or adjuvant only (control) and used to infect HepG2hNTCP cells at 100 GEq/cell. Cell pre‐treatment with Myrcludex B, a highly selective inhibitor of HBV entry, was included in the experiment, as control. HBV infection was further monitored by quantification of secreted HBeAg via ELISA and normalized against levels obtained in the presence of pre‐immune sera. A strong virus neutralization activity against both WT (Figure 7a) and the VEM variant, G145R HBV (Figure 7b) was observed for the immune sera regardless of the plant system used to produce the immunizing antigen, as expected for this chimeric antigen (Pantazica et al., 2022). However, the stronger humoral immune response induced by the FX‐KO N. benthamiana‐produced S/preS116‐42 resulted in significantly higher levels of protection against either the WT HBV or VEM variant. Together, these results demonstrate a better presentation to the immune system or exposure of relevant, virus neutralizing epitopes by the antigen produced in FX‐KO plants, contributing to the observed improved immunogenicity.

The unprecedented global health crisis caused by the COVID‐19 pandemic and leading to the loss of more than 6 million lives since the outbreak in 2019, has shown the devastating consequences of the world's lack of alertness and preparation for emerging viral infections. To prevent epidemics of similar gravity occurring in the future, health care systems must adapt and be able to react promptly by providing efficient diagnostic tools and therapeutic strategies to fight novel infections in a timely manner. The challenges with responding to the increased demand for diverse, high‐quality recombinant viral proteins as vaccines can be addressed by using plants as highly versatile and cost‐efficient protein‐manufacturing platforms. The successful expression of the S‐HBsAg in tobacco plants marked the first report of a plant‐made subunit vaccine and a breakthrough in molecular farming (Mason et al., 1992). Since then, research in plant vaccine production has mainly focused on exploring improved plant transformation technologies and molecular biology approaches to increase recombinant protein yields and recovery. A remarkable diversity of plant species suitable for protein expression in a stable or transient manner and a panel of novel vectors with superior transformation activity, promoters and regulatory sequences, have emerged from these studies (Schillberg and Finnern, 2021). However, in contrast to plantibodies and plant‐produced cytokines, little attention has been paid to potential consequences of “nonhuman” N‐ glycosylation for the immunogenicity of expressed antigens and vaccine efficiency. Rather, the possibility that plant‐specific α‐1,3‐fucose‐ and β‐1,2‐xylose‐linked glycans could trigger adverse effects in humans receiving plant‐derived biopharmaceuticals has been a subject of concern and intense scrutiny (Bosch and Schots, 2010). With the recent technological advances in plant N‐glyco‐engineering, plants devoid of specific xylosyltransferase and fucosyltransferase activities have been developed and are now available for production of recombinant proteins with human‐like N‐glycosylation (Göritzer et al., 2022; Jansing et al., 2019; Strasser et al., 2008). The newly generated hosts are beginning to prove their utility as advanced platforms for expression of therapeutic proteins, as recently demonstrated by the production of therapeutic HIV monoclonal antibodies with improved binding to human Fc receptors (Stelter et al., 2020). Antigen N‐glycosylation may greatly influence the quality of the immune response at several levels. Professional antigen‐presenting cells (APC) express pattern‐recognition receptors (PRRs) on their surface, such as C‐type lectin receptors (CLRs) or Toll‐like receptors (TLRs), involved in recognition and internalization of pathogens or pathogen‐derived molecules (Figdor et al., 2002). N‐linked glycans directly bind to CLRs, thus acting as mediators of glycoantigen uptake; they may also indirectly affect TLRs recognition of pathogens by steric hindrance. Following endocytosis and proteolytic processing of the antigen within the endo‐lysosomal system, generated peptides are loaded onto MHC class II molecules for presentation to naive T cells and initiation of immune signalling (Guermonprez et al., 2002). The presence of N‐glycans can interfere with these processes, influencing the repertoire of generated T cell epitopes. Moreover, it has been shown that glycopeptides can be presented by MHC class II complexes and stimulate specific T‐cell responses. Although binding to the MHC class II occurs through the peptide backbone, the N‐glycan may become the dominant structural element of the epitope recognized by T cells, depending on its location with respect to the peptide‐binding key residues (Werdelin et al., 2002). In this study we sought to investigate the influence of plant‐characteristic glycosylation on the immunogenic properties of the S/preS116‐42 antigen, a newly developed vaccine candidate derived from the S‐HBsAg (Pantazica et al., 2022). Glycosylation of the S‐HBsAg is particularly intriguing, with about half of the synthesized molecules carrying one single N‐glycan at position N146 within the immunodominant region of the protein (Bruss, 2007), likely protecting it from recognition by neutralizing antibodies (Julithe et al., 2014). We found that this glycosylation pattern was preserved in the plant‐expressed S/preS116‐42, regardless of the genetic manipulation of the N‐glycosylation pathway, although the level of the glycosylated isoform was somewhat lower than that produced in mammalian cells. The antigen yields were similar in WT and FX‐KO N. benthamiana plants and significantly improved in comparison with previous studies reporting transient or stable expression of HBsAg in various plants (Pniewski, 2013). Interestingly, BiP expression was up‐regulated in S/preS116‐42‐expressing plants, possibly reflecting the ER stress induced by antigen overload and the rapid cellular response to cope with the folding of a large amount of recombinant protein (Urade, 2007). The antigenic properties of the chimeric protein produced in WT and FX‐KO N. benthamiana plants were remarkably different. Prevention of α‐1,3‐fucosylation and β‐1,2‐xylosylation resulted in enhanced binding to a panel of conformation‐dependent anti‐S antibodies, approaching the recognition of the mammalian cell‐derived antigen. This behaviour may be explained by the N146 glycan being an intrinsic part of a presented MHC class II epitope and hence, its recognition by corresponding antibodies would reflect host‐dependent N‐glycosylation. Alternatively, addition of α‐1,3‐fucose and β‐1,2‐xylose may enhance the epitope shielding effect attributed to the N‐glycan, as described previously (Julithe et al., 2014). Immunization with S/preS116‐42 obtained in FX‐KO N. benthamiana triggered significantly higher antibody titers in mice, with more potent neutralizing activity against native HBV particles, as compared with the antigen produced in WT plants. A similar behaviour was observed during infection with G145R HBV particles, a clinically relevant VEM (Zuckerman, 2000), confirming the superiority of this immune response. The different experimental conditions used in animal trials preclude a quantitative comparison of the efficiency of the immune response induced by the chimeric antigen produced in FX‐KO plants in the current study and that previously obtained in mammalian cells (Pantazica et al., 2022). Nevertheless, the strong virus neutralizing activity of antibodies triggered by either antigen against both WT and VEM HBV suggests a similar quality of the immune response. HBV particles secreted from mammalian cells contain partially sialylated complex‐type oligosaccharides (Schmitt et al., 1999) that are absent in either plant‐made antigen, as revealed by the MS analysis of the N‐linked glycans. Therefore, it is unlikely that the partial “humanized” glycosylation would create novel, better HBV neutralizing glycoepitopes; rather, the absence of α‐1,3‐fucose and β‐1,2‐xylose from the N‐glycan may improve antigen presentation by APCs and/or recognition of peptide–MHC class II complexes by T cell receptors. This hypothesis is supported by the increased levels of IgG2a antibodies induced by the FX‐KO N. benthamiana‐made antigen in vaccinated mice, together with IFN‐γ secretion, indicative of Th1 signalling, activating the cellular arm of the immune response. However, the humoral immune response still remains predominant, as marked by the strong induction of IgG1 and IL‐5. Together, our results highlight in premiere the importance of plant glycosylation in relation to the immunogenicity of protein subunit vaccines and illustrate the potential of FX‐KO N. benthamiana as a highly promising platform for cost‐efficient production of improved HBV antigens. As most prophylactic vaccine candidates against viral infections are highly glycosylated envelope proteins, it will be crucial to investigate in future studies the applicability of this concept to the production of other glycoantigens in plants with “humanized” N‐glycosylation. Moreover, the development of fully “humanized” plant platforms by knocking in mammalian cell‐specific enzymes involved in N‐glycan processing along the secretory pathway remains an important desiderate.

HEK293T and Huh7 cell lines were maintained in Dulbecco's Modified Medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and 1% non‐essential amino acids (NEAA). HepG2hNTCP cells (gift from Professor Stephan Urban, German Center for Infection Research, University of Heidelberg) were cultured and used for HBV neutralization assays as described before (Pantazica et al., 2022). HepG2.2.2.15 cells stably transfected with two copies of the HBV genome (gift from Dr. David Durantel, INSERM U871, Lyon, France), were used for production of WT‐HBV stocks, as previously reported (Dorobantu et al., 2011). All cell lines were maintained at 37 °C, in an incubator supplied with 5% CO2. Stocks of WT HBV and of a clinically relevant VEM, containing the G145R mutation within the antigenic domain of S were also obtained by transfection of Huh7 cells with pGEM‐4Z‐HBV 1.3 WT (gift from Wang‐Shick Ryu, Addgene, #65459) and pGEM‐4Z‐HBV 1.3 G145R (Pantazica et al., 2022), respectively, by using Lipofectamine 3000 (Invitrogen) and following the manufacturer's protocols. Cell media were collected between days 7 and 12 post‐transfection, concentrated by ultracentrifugation on a 20% sucrose layer and quantified by real‐time qPCR (Lazar et al., 2007). HEK 293 T cells were transfected with a pCi plasmid encoding for the S/preS116‐42 antigen, as described (Pantazica et al., 2022).

The sequence coding for HBV S/preS116‐42 was cloned into the plant‐specific expression vector pEAQ‐HT‐DEST1 (Sainsbury et al., 2009). After confirming the successful cloning by sequencing, the vectors were transformed into Agrobacterium tumefaciens LBA4404 cells (Invitrogen), by electroporation. Following cultivation on LB medium containing 50 mg/L kanamycin, for 72 h at 28 °C, a selected transiently transformed strain was used in vacuum‐based agroinfiltration of WT and FX‐KO N. benthamiana, using our in house vacuum setup, as previously described (Dobrica et al., 2021b). Infiltrated plants were grown under standard greenhouse conditions. Expression of the HBV antigen in plant leaves harvested at days 6, 7, 8 and 11 post‐infiltration was determined following protein extraction and western blot. WT N. benthamiana leaves infiltrated with the empty vector pEAQ‐HT‐DEST1 were used as a negative control.

WT and FX‐KO N. benthamiana leaves expressing the S/preS116‐42 antigen or vector‐infiltrated (control) were ground in liquid nitrogen and homogenized in 5 volumes of lysis buffer (0.15 m NaCl, 20 mm Na2HPO4, 20 mm sodium ascorbate, 0.1% Triton X‐100, pH 7) containing a cocktail of protease inhibitors (Santa Cruz Biotechnology) for 30 min, on ice. Plant lysates were centrifuged for 5 min at 1000 × g, 4 °C and the supernatant was subjected to centrifugation at 27000 × g for 15 min at 4 °C. Clear supernatants were collected and 20‐fold concentrated by ultracentrifugation on a 20% sucrose‐bed, for 5 h at 30000 rpm (SW 32 Ti, Beckman Coulter). Samples were further transferred onto a sucrose gradient, consisting of 2 mL layers of 15, 25, 35, 45 and 60% sucrose, respectively and subjected to ultracentrifugation at 32000 rpm (SW 42Ti, Beckman Coulter) for 16 h. The presence of HBV antigens in collected fractions was determined by ELISA, using the Monolisa HBsAg Ultra kit (BioRad). Positive fractions were pooled, dialysed against PBS and incubated with 1% activated carbon (Carbocit®, S.C. Biofarm S.A.) for 4 h at 4 °C with gentle agitation, to remove smaller protein impurities. Protein samples were centrifuged at 10000 × g for 15 min at 4 °C and filtered using a 0.2 μm filter (Millipore). Clear filtrates were further purified via size‐exclusion chromatography on the CaptoCore 400 resin (Cytiva), by gravitational flow. Flow‐through fractions containing the viral antigens were identified as above, pooled and SpeedVac‐concentrated (Thermo Scientific‐Pierce). The total protein content in purified samples was determined by using the BCA assay (Thermo Scientific‐Pierce) and the antigen was quantified by western blot, as previously described (Dobrica et al., 2017).

Protein samples were heat‐denatured by boiling for 10 min in Laemmli buffer in the absence (non‐reducing conditions) or presence (reducing conditions) of 100 mM DTT. Proteins were separated by sodium dodecyl‐sulfate 12% polyacrylamide gel electrophoresis (SDS‐PAGE) and either Coomassie‐stained (ReadyBlue™ Protein Gel Stain, Sigma‐Aldrich) or transferred onto nitrocellulose membranes. Following treatment with 10% skimmed dry milk in PBS for 1 h, membranes were sequentially incubated with primary antibodies: either mouse anti‐preS1 monoclonal antibodies (sc‐57 762, Santa Cruz Biotechnology, 1:1000) or rabbit anti‐S polyclonal antibodies (NB100‐62652, Novus Biologicals, 1:1000), mouse anti‐BiP monoclonal antibodies (sc‐376 768, Santa Cruz Biotechnology, 1:300) or mouse, HRP conjugated anti‐GAPDH polyclonal antibodies (sc‐25 778, Santa Cruz Biotechnology, 1:500) overnight at 4 °C, followed by HRP‐conjugated mouse‐IgGκ binding protein (sc‐516 102, Santa Cruz Biotechnology, 1:10000) or HRP‐conjugated anti‐rabbit polyclonal antibodies (sc‐2357, Santa Cruz Biotechnology, 1:10000), respectively, for 1 h at room temperature (RT). Proteins were visualized by using an enhanced chemiluminescence assay (ECL, Thermo Scientific‐Pierce). Protein densitometry was carried out by using the Image J software (National Institutes of Health) and antigen concentrations were estimated by using a standard curve of commercial L protein processed under the same experimental conditions (preS1 antigen, Beacle).

The analysis was performed by Asparia Glycomics (Donostia‐San Sebastian, Spain), as briefly described below. The bands corresponding to the monomeric antigen were excised from Coomassie‐stained gels. The gel pieces were destained by treatment with 100 mM ammonium bicarbonate/acetonitrile (1:1), overnight, followed by extensive washing with ultrapure water and incubation with acetonitrile until complete removal of the stain. The pieces were SpeedVac‐dried then treated with 1 mL of reducing buffer (10 mM DTT, 100 mm ammonium bicarbonate) at 56 °C for 30 min. The reducing buffer was discarded and 500 μL of alkylating buffer (55 mm iodoacetamide, 100 mm ammonium bicarbonate) was added to the samples for 30 min, at RT, in the dark. The gel pieces were extensively washed with ultrapure water followed by acetonitrile then SpeedVac‐dried at RT. For de‐glycosylation, 25 μL of the PNGase A enzyme solution (150 mU/50 μL, New England Biolabs) were added to the dried gel pieces for 5 min at RT, followed by addition of 50 mM ammonium bicarbonate and overnight incubation at 37 °C. Samples were then centrifuged at 12000 × g for 5 min and the incubation buffer was discarded. Next, 1 mL of ultrapure water was added to the samples and sonicated for 30 min for glycan extraction by using an Emmi‐H30 water ultrasonic bath (EMAG, Germany). This process was repeated twice, with a sonication time of 1 h for the last run. The water extractions were collected, lyophilized and reconstituted in 10 μL of ultrapure water. Released glycans were treated with 50 μL of procainamide labeling solution (12 mg of procainamide, 6 mg of sodium cyanoborohydride in 0.5 mL of a dimethyl sulfoxide/acetic acid, 7/3 mixture), for 3 h at 65 °C. Labelled glycan samples were diluted with 200 μL acetonitrile and directly analysed by UPLC‐FLD/MS (Waters, Milford, MA), under standard conditions (Waters, Application Note UPLC/FLR/QTof MS Analysis of Procainamide‐Labelled N‐glycans). Labelled glycan peaks were assigned to glycan structures based on mass, as determined by the electrospray ionization (ESI)‐MS spectrum at their corresponding retention time. For quantification, peak areas were integrated and converted into relative percentages of the total glycan amount, defined as the sum of all peaks and normalized to 100%.

To determine the antigenicity of HBV S/preS116‐42 produced in different host systems, 96‐well MaxiSorp plates (Nunc Thermo Scientific) were coated with 50 ng/well of various conformational anti‐S antibodies raised in mice, namely #10‐H05G, #10‐H05H, #10–1323, #10–1324 and #10‐H05M (Fitzgerald Industries International), overnight at 4 °C. Subsequently, the plates were washed and blocked in 10% dried skim milk in PBS for 2 h. The plates were then incubated with serial dilutions (1:20 to 1:25600) of HEK293T cells or N. benthamiana lysates containing equal amounts of the HBV S/preS116‐42 antigen, as quantified via western blot using anti‐preS1 antibodies (Dobrica et al., 2017). The plates were washed extensively with PBS supplemented with 0.1% Tween‐20 (Sigma Aldrich) and incubated with a conformation‐independent, rabbit anti‐S antibody (NB100‐62652, Novus Biologicals, 1:1000) for 1 h at 37 °C. This antibody was shown to bind the chimeric HBV antigen with similar efficiency, regardless of the production host (Figure S1). After another round of extensive washing, the plates were incubated with HRP‐conjugated anti‐rabbit antibodies (sc‐2357, Santa Cruz Biotechnology, 1:10000) for 1 h at RT and then washed 5 times with PBS supplemented with 0.1% Tween‐20. Detection was performed by incubation with the 3,3′,5,5′‐Tetramethylbenzidine (TMB) substrate (BioRad) for 30 min, after which the enzymatic reaction was stopped via addition of 2 N H2SO4. The OD was read at 450 nm in a Mithras Microplate Reader.

Animal experiments were conducted in accordance with standards set forth in the Council Directive 86/609/EEC and national legislation. The study was approved by the Ethical Committee of the “Cantacuzino” Medico‐Military National Research Institute, number 374/10.12.2019 and the national designated authority, ANSVSA, number 488/22.01.2020. Three groups of female Balb/c mice (6–8 weeks‐old) were immunized by intramuscular injection, three times, at a 14‐day interval. The first two groups (n = 7) received 25 μg of either WT‐ or FX‐KO N. benthamiana‐produced HBV‐S/preS116‐42 antigen combined with 25 μL of AddaVax (InvivoGen, San Diego, CA), as adjuvant, and PBS to a final volume of 50 μL/dose. The third group representing the control lot (n = 5) received the same volume of adjuvant combined with PBS. Blood samples were collected from individual mice by orbital puncture prior to administration of the first dose, and after the first and second boost, at days 28 and 42, respectively. Blood sera were obtained from clotted blood following centrifugation at 14, 000 × g for 10 min at RT, then stored at −80 °C until further use. The sera were stable for at least 3 months, with no loss of activity in multiple experimental runs of antibody titration and analysis of virus neutralizing capacity. The animals were sacrificed on day 49 under anaesthesia after the final bleed and spleens were removed aseptically for analysis of the cellular immune response.

The antigen specific IgG and IgG1, IgG2a subclasses were detected by ELISA as previously described (Pantazica et al., 2022), by using a suspension of UV‐inactivated HBV in PBS (containing 0.4 μg/mL of HBsAg) for plate coating.

Antigen‐specific IFN‐γ and IL‐5 secreting cells were quantified by ELISpot, using the Mouse IFN‐γ/IL‐5 FluoroSpot kit (Mabtech, Sweden). Briefly, plates were coated with 100 μL of 15 μg/mL anti‐IFN‐γ and anti‐IL‐5 capture antibodies, overnight at 4 °C. Plates were extensively washed (5 times) in sterile PBS and incubated for 2 h with 1% bovine serum albumin (BSA) in PBS, to block nonspecific interactions. Splenocytes isolated from immunized mice were seeded in plates at 106 and 105 cells/well, in RPMI‐1640 containing 10% fetal calf serum (FCS) and 2‐mercaptoethanol. Triplicate samples were stimulated with UV‐inactivated HBV (5 μg/mL HBsAg), CD3/CD28 (0.5 μg/mL), ConA (5 μg/mL) or PBS for 36 h at 37 °C, in a 5% CO2 incubator. Cells were then removed and plates were washed 5 times with PBS before addition of 100 μL/well of detection antibody solution (anti‐IFN‐γ‐FITC and anti‐IL‐5‐biotin, diluted 1:200 and 1:500, respectively, in PBS with 0.1% BSA). Plates were further incubated for 2 h at RT and then washed with PBS as above. Detection was performed by treatment with anti‐FITC‐green (1:100) and Streptavidin‐red (1:200) for 1 h at RT, in the dark. The plates were washed again with PBS, followed by 15 min incubation with Fluorescence enhancer‐II and drying in the dark. Fluorescence photographs, using the appropriate filters were acquired on an AID iSpot FluoroSpot Reader and spot detection and counting was performed using the AID EliSpot software Version 7.0. Comparison between groups and statistical analysis was performed by using the Wilcoxon rank‐sum test.

HepG2hNTCP cells were seeded in 48‐well plates 1 day prior to HBV infection. Sera from mice immunized with adjuvant or the plant‐produced HBV‐S/preS116‐42 antigen were collected at day 49 post‐immunization and diluted 1:50 in complete DMEM media supplemented with 4% polyethylene glycol (PEG, Sigma Aldrich). Pre‐immune sera from individual mice were pooled per mice group and diluted in the same manner. WT HBV and the vaccine escape HBV‐SG145R (100 genome equivalents/cell) were incubated with diluted sera for 1 h at 37 °C, before infection. Control conditions included cell treatment with 1 μm Myrcludex B (Pepscan), a well‐established inhibitor of HBV entry (Urban et al., 2014), for 3 h prior to virus inoculation and cells infected with untreated HBV. At 16 h post‐infection, cells were extensively washed with PBS and incubated with complete DMEM media, supplemented with 2.5% DMSO (AppliChem). The medium collected between days 7–11 post‐infection was retained for quantification of the HBeAg by using the Monolisa HBe Ag‐Ab PLUS kit (BioRad). Inhibition of HBV infection by the immune sera was represented as percentage from infectivity values in the presence of the pre‐immune sera, at the same dilution. Data were statistically analysed by using the Mann–Whitney U test in GraphPad Prism version 6.

N.B.N., J.L.C. and C.S. conceived the study and analysed the data. A‐M.P., M.O.D and C.L obtained the HBV construct for mammalian cell expression. A‐M.P. performed antigen extraction, purification and molecular characterization, obtained the WT‐HBV and VEM stocks and carried out virus neutralization studies. M.O.D. performed antigen extraction and quantification and the ER stress analysis. J.L.C, A.E. and H.S. made the HBV DNA constructs for plant expression and obtained the antigen in WT and FX‐KO N. benthamiana. C.S. and A.O. conceived animal experiments. I.C., C.T., I.I. A.C. and C.S. performed the animal experiments. C.S and A.O. analysed the immunology data. N.B.N, A‐M.P., M.O.D, J.L.C., and C.S. wrote the manuscript.

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