Plant-made vaccines against viral diseases in humans and farm animals.

Plants provide not only food and feed, but also herbal medicines and various raw materials for industry. Moreover, plants can be green factories producing high value bioproducts such as biopharmaceuticals and vaccines. Advantages of plant-based production platforms include easy scale-up, cost effectiveness, and high safety as plants are not hosts for human and animal pathogens. Plant cells perform many post-translational modifications that are present in humans and animals and can be essential for biological activity of produced recombinant proteins. Stimulated by progress in plant transformation technologies, substantial efforts have been made in both the public and the private sectors to develop plant-based vaccine production platforms. Recent promising examples include plant-made vaccines against COVID-19 and Ebola. The COVIFENZ® COVID-19 vaccine produced in Nicotiana benthamiana has been approved in Canada, and several plant-made influenza vaccines have undergone clinical trials. In this review, we discuss the status of vaccine production in plants and the state of the art in downstream processing according to good manufacturing practice (GMP). We discuss different production approaches, including stable transgenic plants and transient expression technologies, and review selected applications in the area of human and veterinary vaccines. We also highlight specific challenges associated with viral vaccine production for different target organisms, including lower vertebrates (e.g., farmed fish), and discuss future perspectives for the field.

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 ß-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 ß-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.

Efficient cellular and humoral immune response and production of virus-neutralizing antibodies by the Hepatitis B Virus S/preS116-42 antigen.

Despite the availability of improved antiviral therapies, infection with Hepatitis B virus (HBV) remains a3 significant health issue, as a curable treatment is yet to be discovered. Current HBV vaccines relaying on the efficient expression of the small (S) envelope protein in yeast and the implementation of mass vaccination programs have clearly contributed to containment of the disease. However, the lack of an efficient immune response in up to 10% of vaccinated adults, the controversies regarding the seroprotection persistence in vaccine responders and the emergence of vaccine escape virus mutations urge for the development of better HBV immunogens. Due to the critical role played by the preS1 domain of the large (L) envelope protein in HBV infection and its ability to trigger virus neutralizing antibodies, including this protein in novel vaccine formulations has been considered a promising strategy to overcome the limitations of S only-based vaccines. In this work we aimed to combine relevant L and S epitopes in chimeric antigens, by inserting preS1 sequences within the external antigenic loop of S, followed by production in mammalian cells and detailed analysis of their antigenic and immunogenic properties. Of the newly designed antigens, the S/preS116-42 protein assembled in subviral particles (SVP) showed the highest expression and secretion levels, therefore, it was selected for further studies in vivo. Analysis of the immune response induced in mice vaccinated with S/preS116-42- and S-SVPs, respectively, demonstrated enhanced immunogenicity of the former and its ability to activate both humoral and cellular immune responses. This combined activation resulted in production of neutralizing antibodies against both wild-type and vaccine-escape HBV variants. Our results validate the design of chimeric HBV antigens and promote the novel S/preS1 protein as a potential vaccine candidate for administration in poor-responders to current HBV vaccines.

Probing the Hepatitis B Virus E-Antigen with a Nanopore Sensor Based on Collisional Events Analysis.

Real-time monitoring, simple operation, and cheaper methods for detecting immunological proteins hold the potential for a solid influence on proteomics and human biology, as they can promote the onset of timely diagnoses and adequate treatment protocols. In this work we present an exploratory study suggesting the applicability of resistive-pulse sensing technology in conjunction with the α-hemolysin (α-HL) protein nanopore, for the detection of the chronic hepatitis B virus (HBV) e-antigen (HBeAg). In this approach, the recognition between HBeAg and a purified monoclonal hepatitis B e antibody (Ab(HBeAg)) was detected via transient ionic current spikes generated by partial occlusions of the α-HL nanopore by protein aggregates electrophoretically driven toward the nanopore's vestibule entrance. Despite the steric hindrance precluding antigen, antibody, or antigen-antibody complex capture inside the nanopore, their stochastic bumping with the nanopore generated clear transient blockade events. The subsequent analysis suggested the detection of protein subpopulations in solution, rendering the approach a potentially valuable label-free platform for the sensitive, submicromolar-scale screening of HBeAg targets.

Sac1 phosphatidylinositol 4-phosphate phosphatase is a novel host cell factor regulating hepatitis B virus particles assembly and release.

The life-cycle of the Hepatitis B Virus (HBV), an enveloped DNA virus affecting the lives of more than 296 million chronicallyinfected people, is tightly dependent on the lipid metabolism of the host cell. Fatty acids and cholesterol are among the lipid factors with documented roles in regulating HBV replication and infection, respectively, but little is known about the phosphoinositide metabolism in these processes. In this study, we investigated the role of Sac1, a highly conserved phosphatidylinositol-4-phosphate (PI4P) phosphatase, with essential functions in phospholipid metabolism, in HBV assembly, and release. PI4P is one of the most abundant cellular phosphoinositide with complex functions at the level of the secretory pathway. Owing to the highly specific phosphatase activity toward PI4P, Sac1 controls the levels and restricts the localization of this lipid particularly at the trans-Golgi network, where it regulates sphingolipid synthesis, proteins sorting, and vesicles budding, by recruiting specific adaptor proteins. As a complete loss of Sac1 function compromises cell viability, in this work, we first developed and characterized several HBV replication-permissive cellular models with a moderate, transient, or stable downregulation of Sac1 expression. Our results show that Sac1 depletion in hepatic cells results in increased levels and redistribution of intracellular PI4P pools and impaired trafficking of the HBV envelope proteins to the endosomal vesicular network. Importantly, virus envelopment and release from these cells are significantly inhibited, revealing novel roles for Sac1, as a key host cell factor regulating morphogenesis of a DNA virus.

Challenges and Prospects of Plant-Derived Oral Vaccines against Hepatitis B and C Viruses.

Hepatitis B and C viruses chronically affect approximately 3.5% of the global population, causing more than 800,000 deaths yearly due to severe liver pathogenesis. Current HBV vaccines have significantly contributed to the reduction of chronic HBV infections, supporting the notion that virus eradication is a feasible public health objective in the near future. In contrast to HBV, a prophylactic vaccine against HCV infection is not available yet; however, intense research efforts within the last decade have significantly advanced the field and several vaccine candidates are shortlisted for clinical trials. A successful vaccine against an infectious disease of global importance must not only be efficient and safe, but also easy to produce, distribute, administer, and economically affordable to ensure appropriate coverage. Some of these requirements could be fulfilled by oral vaccines that could complement traditional immunization strategies. In this review, we discuss the potential of edible plant-based oral vaccines in assisting the worldwide fight against hepatitis B and C infections. We highlight the latest research efforts to reveal the potential of oral vaccines, discuss novel antigen designs and delivery strategies, as well as the limitations and controversies of oral administration that remain to be addressed to make this approach successful.

Hepatitis C virus E2 envelope glycoprotein produced in Nicotiana benthamiana triggers humoral response with virus-neutralizing activity in vaccinated mice.

Chronic infection with hepatitis C virus (HCV) remains a leading cause of liver-related pathologies and a global health problem, currently affecting more than 71 million people worldwide. The development of a prophylactic vaccine is much needed to complement the effective antiviral treatment available and achieve HCV eradication. Current strategies focus on increasing the immunogenicity of the HCV envelope glycoprotein E2, the major target of virus-neutralizing antibodies, by testing various expression systems or manipulating the protein conformation and the N-glycosylation pattern. Here we report the first evidence of successful production of the full-length HCV E2 glycoprotein in Nicotiana benthamiana, by using the Agrobacterium-mediated transient expression technology. Molecular and functional analysis showed that the viral protein was correctly processed in plant cells and achieved the native folding required for binding to CD81, one of the HCV receptors. N-glycan analysis of HCV-E2 produced in N. benthamiana and mammalian cells indicated host-specific trimming of mannose residues and possibly, protein trafficking. Notably, the plant-derived viral antigen triggered a significant immune response in vaccinated mice, characterized by the presence of antibodies with HCV-neutralizing activity. Together, our study demonstrates that N. benthamiana is a viable alternative to costly mammalian cell cultures for the expression of complex viral antigens and supports the use of plants as cost-effective production platforms for the development of HCV vaccines.

Production of Chimeric Hepatitis B Virus Surface Antigens in Mammalian Cells.

The small (S) envelope protein of the Hepatitis B Virus (HBV), HBV-S, has the unique ability to self-assemble into highly immunogenic subviral particles (SVPs), in the absence of other viral factors, in eukaryotic cells, including those of nonhepatic origin. This feature is currently exploited for generation of SVPs exposing heterologous epitopes on their surface that can be used as vaccine candidates to target various diseases. Here, we describe a simple and robust method for production of such chimeric HBV-S protein-based SVPs in transiently transfected HEK293T cells and purification from cell supernatants by ultracentrifugation on sucrose cushion and sucrose step gradients. The SVPs obtained by this methodology have been successfully used in immunogenicity studies in animal models.

N-Glycosylation and N-Glycan Processing in HBV Biology and Pathogenesis.

Hepatitis B Virus (HBV) glycobiology has been an area of intensive research in the last decades and continues to be an attractive topic due to the multiple roles that N-glycosylation in particular plays in the virus life-cycle and its interaction with the host that are still being discovered. The three HBV envelope glycoproteins, small (S), medium (M) and large (L) share a very peculiar N-glycosylation pattern, which distinctly regulates their folding, degradation, assembly, intracellular trafficking and antigenic properties. In addition, recent findings indicate important roles of N-linked oligosaccharides in viral pathogenesis and evasion of the immune system surveillance. This review focuses on N-glycosylation's contribution to HBV infection and disease, with implications for development of improved vaccines and antiviral therapies.

Oral administration of a chimeric Hepatitis B Virus S/preS1 antigen produced in lettuce triggers infection neutralizing antibodies in mice.

Hepatitis B Virus (HBV) infection can be prevented by vaccination. Vaccines containing the small (S) envelope protein are currently used in universal vaccination programs and achieve protective immune response in more than 90% of recipients. However, new vaccination strategies are necessary for successful immunization of the remaining non- or low-responders. We have previously characterized a novel HBV chimeric antigen, which combines neutralization epitopes of the S and the preS1 domain of the large (L) envelope protein (genotype D). The S/preS121-47 chimera produced in mammalian cells and Nicotiana benthamiana plants, induced a significantly stronger immune response in parenterally vaccinated mice than the S protein. Here we describe the transient expression of the S/preS121-47 antigen in an edible plant, Lactuca sativa, for potential development of an oral HBV vaccine. Our study shows that oral administration of adjuvant-free Lactuca sativa expressing the S/preS121-47 antigen, three times, at 1 µg/dose, was sufficient to trigger a humoral immune response in mice. Importantly, the elicited antibodies were able to neutralize HBV infection in an NTCP-expressing infection system (HepG2-NTCP cell line) more efficiently than those induced by mice fed on Lactuca sativa expressing the S protein. These results support the S/preS121-47 antigen as a promising candidate for future development as an edible HBV vaccine.

A novel chimeric Hepatitis B virus S/preS1 antigen produced in mammalian and plant cells elicits stronger humoral and cellular immune response than the standard vaccine-constituent, S protein.

Chronic Hepatitis B Virus (HBV) infection leads to severe liver pathogenesis associated with significant morbidity and mortality. As no curable medication is yet available, vaccination remains the most cost-effective approach to limit HBV spreading and control the infection. Although safe and efficient, the standard vaccine based on production of the small (S) envelope protein in yeast fails to elicit an effective immune response in about 10% of vaccinated individuals, which are at risk of infection. One strategy to address this issue is the development of more immunogenic antigens. Here we describe a novel HBV antigen obtained by combining relevant immunogenic determinants of S and large (L) envelope proteins. Our approach was based on the insertion of residues 21-47 of the preS1 domain of the L protein (nomenclature according to genotype D), involved in virus attachment to hepatocytes, within the external antigenic loop of S. The resulting S/preS121-47 chimera was successfully produced in HEK293T and Nicotiana benthamiana plants, as a more economical recombinant protein production platform. Comparative biochemical, functional and electron microscopy analysis indicated assembly of the novel antigen into subviral particles in mammalian and plant cells. Importantly, these particles preserve both S- and preS1-specific epitopes and elicit significantly stronger humoral and cellular immune responses than the S protein, in both expression systems used. Our data promote this antigen as a promising vaccine candidate to overcome poor responsiveness to the conventional, S protein-based, HBV vaccine.

Lettuce-produced hepatitis C virus E1E2 heterodimer triggers immune responses in mice and antibody production after oral vaccination.

The hepatitis C virus (HCV) is a major etiologic agent for severe liver diseases (e.g. cirrhosis, fibrosis and hepatocellular carcinoma). Approximately 140 million people have chronic HCV infections and about 500 000 die yearly from HCV-related liver pathologies. To date, there is no licensed vaccine available to prevent HCV infection and production of a HCV vaccine remains a major challenge. Here, we report the successful production of the HCV E1E2 heterodimer, an important vaccine candidate, in an edible crop (lettuce, Lactuca sativa) using Agrobacterium-mediated transient expression technology. The wild-type dimer (E1E2) and a variant without an N-glycosylation site in the E2 polypeptide (E1E2∆N6) were expressed, and appropriate N-glycosylation pattern and functionality of the E1E2 dimers were demonstrated. The humoral immune response induced by the HCV proteins was investigated in mice following oral administration of lettuce antigens with or without previous intramuscular prime with the mammalian HEK293T cell-expressed HCV dimer. Immunization by oral feeding only resulted in development of weak serum levels of anti-HCV IgM for both antigens; however, the E1E2∆N6 proteins produced higher amounts of secretory IgA, suggesting improved immunogenic properties of the N-glycosylation mutant. The mice group receiving the intramuscular injection followed by two oral boosts with the lettuce E1E2 dimer developed a systemic but also a mucosal immune response, as demonstrated by the presence of anti-HCV secretory IgA in faeces extracts. In summary, our study demonstrates the feasibility of producing complex viral antigens in lettuce, using plant transient expression technology, with great potential for future low-cost oral vaccine development.

Development of a DsRed-expressing HepaRG cell line for real-time monitoring of hepatocyte-like cell differentiation by fluorescence imaging, with application in screening of novel geometric microstructured cell growth substrates.

The bipotent nature of the HepaRG cell line is a unique property among human hepatoma-derived cells. Cell treatment with specific differentiation inducers results in a mixture of hepatocyte- and biliary-like cells, accompanied by upregulation of liver-specific proteins, drug metabolizing enzymes, transcription regulators, membrane receptors or innate immune response effectors. These features make the HepaRG cells a suitable and handy replacement for primary hepatocytes, to study hepatic functions in vitro. However, cell differentiation is a long, variable process, requiring special culture conditions, while the resulting mixed cell populations is usually a major drawback. This process can potentially be controlled by interface characteristics, such as substrate topography. To screen for such novel substrates, we have first developed a new HepaRG cell line, designated as HepaRGDsRed, expressing the reporter gene DsRed. The fluorescent protein was expressed in hepatocyte- and not biliary-like cells, in a differentiation dependent-manner. We have further used replicated microstructured gradients of polydimethylsiloxane (PDMS) that allow three-dimensional manipulation in vitro, to monitor HepaRGDsRed differentiation in real time. We demonstrate that this approach enables the controlled assembly of viable hepatocyte-like cells for functional studies, which can be maintained in culture without loss of differentiation. The regulated expression of the DsRed reporter proved a valuable tool not only for rapid screening of novel cell growth substrates favoring cell differentiation, but also, to enrich the hepatocyte-like cell population by fluorescence-activated cell sorting to investigate liver-specific processes in vitro.

Novel function of the endoplasmic reticulum degradation-enhancing α-mannosidase-like proteins in the human hepatitis B virus life cycle, mediated by the middle envelope protein.

Cells replicating the human hepatitis B virus (HBV) express high levels of degradation-enhancing α-mannosidase-like proteins (EDEMs), a family of proteins involved in the endoplasmic reticulum associated degradation, one of the pathways activated during the unfolded protein response. Owing to their α-1,2 mannosidase activity, the EDEM1-3 proteins are able to process the N-linked glycans of misfolded or incompletely folded proteins, providing the recognition signal for their subsequent degradation. The HBV small (S), medium (M), and large (L) surface proteins bear an N-linked glycosylation site in the common S domain that is partially occupied in all proteins. The M protein contains an additional site in its preS2 domain, which is always functional. Here, we report that these oligosaccharides are processed by EDEMs, more efficiently by EDEM3, which induces degradation of L and S proteins, accompanied by a reduction of subviral particles production. In striking contrast, M not only is spared from degradation but its trafficking is also accelerated leading to an improved secretion. This unusual behavior of the M protein requires strictly the mannose trimming of the preS2 N-linked glycan. Furthermore, we show that HBV secretion is significantly inhibited under strong endoplasmic reticulum stress conditions when M expression is prevented by mutagenesis of the viral genome. These observations unfold unique properties of the M protein in the HBV life cycle during unfolded protein response and point to alternative mechanisms employed by EDEMs to alleviate this stress in case of necessity by promoting glycoprotein trafficking rather than degradation.

Modulation of the unfolded protein response by the human hepatitis B virus.

During productive viral infection the host cell is confronted with synthesis of a vast amount of viral proteins which must be folded, quality controlled, assembled and secreted, perturbing the normal function of the endoplasmic reticulum (ER). To counteract the ER stress, cells activate specific signaling pathways, designated as the unfolded proteins response (UPR), which essentially increase their folding capacity, arrest protein translation, and degrade the excess of misfolded proteins. This cellular defense mechanism may, in turn, affect significantly the virus life-cycle. This review highlights the current understanding of the mechanisms of the ER stress activation by Human Hepatitis B virus (HBV), a deadly pathogen affecting more than 350 million people worldwide. Further discussion addresses the latest discoveries regarding the adaptive strategies developed by HBV to manipulate the UPR for its own benefits, the controversies in the field and future perspectives.

Using proteomics to unravel the mysterious steps of the HBV-life-cycle.

Infection with Hepatitis B virus (HBV) is the most common cause of liver disease in the world. Infection becomes chronic in up to 10 % of adults, with severe consequences on liver function, including inflammation, fibrosis, cirrhosis, and eventually hepatocellular carcinoma (HCC). HCC is a fast progressing disease causing the death of approximately one million patients annually; current treatment has very limited success, mainly due to late-stage diagnosis and poor screening methodologies. Therefore, unraveling the complex HBV-host cell interactions during progression of the disease is of crucial importance, not only to understand the mechanisms underlying carcinogenesis, but importantly, for the development of new biomarkers for prognostic and early diagnosis. This is an area of research strongly influenced by proteomic studies, which have benefited in the last decade from major technical improvements in accuracy of quantification and sensitivity, large-scale analysis of low-abundant proteins, such as those from clinical samples being now possible and widely applied. This work is a critical review of the impact of the proteomic studies on our current understanding of HBV-associated pathogenesis, diagnostics, and treatment.

Direct observation of athermal photofluidisation in azo-polymer films.

The surface relief gratings (SRGs) can be generated when azo-polymer films are exposed to laser beam interference as a result of mass migration. Despite considerable research effort over the past two decades this complex phenomenon remains incompletely understood. Here we show, for the first time, the athermal photofluidisation of azo-polysiloxane films exposed to 488 nm light, directly monitored by optical microscopy. A process of surface relief erasure occurring in parallel with its inscription was also observed during laser irradiation. We therefore propose a new mechanism of SRG formation, based on three different processes: (1) the polymer photo-fluidization in illuminated regions, (2) the mass displacement from illuminated to dark regions and (3) the inverse mass displacement, from dark to illuminated regions. The mechanical properties of the films during UV light irradiation were investigated by classical rheology and, for the first time, by using amplitude modulation-frequency modulation atomic force microscopy (AM-FM AFM).

Paracrine signals from liver sinusoidal endothelium regulate hepatitis C virus replication.

UNLABELLED: Hepatitis C virus (HCV) is a major cause of global morbidity, causing chronic liver injury that can progress to cirrhosis and hepatocellular carcinoma. The liver is a large and complex organ containing multiple cell types, including hepatocytes, sinusoidal endothelial cells (LSEC), Kupffer cells, and biliary epithelial cells. Hepatocytes are the major reservoir supporting HCV replication; however, the role of nonparenchymal cells in the viral lifecycle remains largely unexplored. LSEC secrete factors that promote HCV infection and transcript analysis identified bone morphogenetic protein 4 (BMP4) as a candidate endothelial-expressed proviral molecule. Recombinant BMP4 increased HCV replication and neutralization of BMP4 abrogated the proviral activity of LSEC-conditioned media. Importantly, BMP4 expression was negatively regulated by vascular endothelial growth factor A (VEGF-A) by way of a VEGF receptor-2 (VEGFR-2) primed activation of p38 MAPK. Consistent with our in vitro observations, we demonstrate that in normal liver VEGFR-2 is activated and BMP4 expression is suppressed. In contrast, in chronic liver disease including HCV infection where there is marked endothelial cell proliferation, we observed reduced endothelial cell VEGFR-2 activation and a concomitant increase in BMP4 expression. CONCLUSION: These studies identify a role for LSEC and BMP4 in HCV infection and highlight BMP4 as a new therapeutic target for treating individuals with liver disease.

Modulation of the antioxidant/pro-oxidant balance, cytotoxicity and antiviral actions of grape seed extracts.

Grape seed extracts (GSEs) were investigated in yeast cells harbouring defects in their antioxidant system (regarding the cellular growth and growth recovery from H2O2 insult). GSEs antioxidant activity was detected in wild-type and mutant strains Δcta1, Δgsh1 and Δoye2glr1, while pro-oxidant activity in Δsod1 cells was seen. Assessment of proliferation of prostate cancer PC3 and HBV-replicating HepG2 2.2.15 cells treated with GSEs has shown higher cytotoxicity of red grape seed extract (RW) than white grape seed extract (WW) subjective to dose and period of administration. No antiviral effect was detected by measuring the secreted virion particles in HepG2 2.2.15 cells treated with GSEs. The GSEs play a dual antioxidant/pro-oxidant role in vivo according with the cellular antioxidant system deficiencies and exhibit cytotoxic properties in PC3 and HepG2 2.2.15 cell lines, but no antiviral action against HBV.