Role of contacts in long-range protein conductance.

Proteins are widely regarded as insulators, despite reports of electrical conductivity. Here we use measurements of single proteins between electrodes, in their natural aqueous environment to show that the factor controlling measured conductance is the nature of the electrical contact to the protein, and that specific ligands make highly selective electrical contacts. Using six proteins that lack known electrochemical activity, and measuring in a potential region where no ion current flows, we find characteristic peaks in the distributions of measured single-molecule conductances. These peaks depend on the contact chemistry, and hence, on the current path through the protein. In consequence, the measured conductance distribution is sensitive to changes in this path caused by ligand binding, as shown with streptavidin-biotin complexes. Measured conductances are on the order of nanosiemens over distances of many nanometers, orders of magnitude more than could be accounted for by electron tunneling. The current is dominated by contact resistance, so the conductance for a given path is independent of the distance between electrodes, as long as the contact points on the protein can span the gap between electrodes. While there is no currently known biological role for high electronic conductance, its dependence on specific contacts has important technological implications, because no current is observed at all without at least one strongly bonded contact, so direct electrical detection is a highly selective and label-free single-molecule detection method. We demonstrate single-molecule, highly specific, label- and background free-electronic detection of IgG antibodies to HIV and Ebola viruses.

In vitro and in vivo efficacy of anti-chikungunya virus monoclonal antibodies produced in wild-type and glycoengineered Nicotiana benthamiana plants.

Chikungunya virus (CHIKV) is a mosquito-transmitted alphavirus, and its infection can cause long-term debilitating arthritis in humans. Currently, there are no licensed vaccines or therapeutics for human use to combat CHIKV infections. In this study, we explored the feasibility of using an anti-CHIKV monoclonal antibody (mAb) produced in wild-type (WT) and glycoengineered (∆XFT) Nicotiana benthamiana plants in treating CHIKV infection in a mouse model. CHIKV mAb was efficiently expressed and assembled in plant leaves and enriched to homogeneity by a simple purification scheme. While mAb produced in ∆XFT carried a single N-glycan species at the Fc domain, namely GnGn structures, WT produced mAb exhibited a mixture of N-glycans including the typical plant GnGnXF3 glycans, accompanied by incompletely processed and oligomannosidic structures. Both WT and ∆XFT plant-produced mAbs demonstrated potent in vitro neutralization activity against CHIKV. Notably, both mAb glycoforms showed in vivo efficacy in a mouse model, with a slight increased efficacy by the ∆XFT-produced mAbs. This is the first report of the efficacy of plant-produced mAbs against CHIKV, which demonstrates the ability of using plants as an effective platform for production of functionally active CHIKV mAbs and implies optimization of in vivo activity by controlling Fc glycosylation.

Plant-produced Zika virus envelope protein elicits neutralizing immune responses that correlate with protective immunity against Zika virus in mice.

The global Zika virus (ZIKV) outbreak and its link to foetal and newborn microcephaly and severe neurological complications in adults call for the urgent development of ZIKV vaccines. In response, we developed a subunit vaccine based on the ZIKV envelope (E) protein and investigated its immunogenicity in mice. Transient expression of ZIKV E (zE) resulted in its rapid accumulation in leaves of Nicotiana benthamiana plants. Biochemical analysis revealed that plant-produced ZIKV E (PzE) exhibited specific binding to a panel of monoclonal antibodies that recognize various zE conformational epitopes. Furthermore, PzE can be purified to >90% homogeneity with a one-step Ni2+ affinity chromatography process. PzE are found to be highly immunogenic, as two doses of PzE elicited both potent zE-specific antibody and cellular immune responses in mice. The delivery of PzE with alum induced a mixed Th1/Th2 immune response, as the antigen-specific IgG isotypes were a mixture of high levels of IgG1/IgG2c and splenocyte cultures from immunized mice secreted significant levels of IFN-gamma, IL-4 and IL-6. Most importantly, the titres of zE-specific and neutralizing antibodies exceeded the threshold that correlates with protective immunity against multiple strains of ZIKV. Thus, our results demonstrated the feasibility of plant-produced ZIKV protein antigen as effective, safe and affordable vaccines against ZIKV.

Development of Antibody Therapeutics against Flaviviruses.

Recent outbreaks of Zika virus (ZIKV) highlight the urgent need to develop efficacious interventions against flaviviruses, many of which cause devastating epidemics around the world. Monoclonal antibodies (mAb) have been at the forefront of treatment for cancer and a wide array of other diseases due to their specificity and potency. While mammalian cell-produced mAbs have shown promise as therapeutic candidates against several flaviviruses, their eventual approval for human application still faces several challenges including their potential risk of predisposing treated patients to more severe secondary infection by a heterologous flavivirus through antibody-dependent enhancement (ADE). The high cost associated with mAb production in mammalian cell cultures also poses a challenge for the feasible application of these drugs to the developing world where the majority of flavivirus infection occurs. Here, we review the current therapeutic mAb candidates against various flaviviruses including West Nile (WNV), Dengue virus (DENV), and ZIKV. The progress of using plants for developing safer and more economical mAb therapeutics against flaviviruses is discussed within the context of their expression, characterization, downstream processing, neutralization, and in vivo efficacy. The progress of using plant glycoengineering to address ADE, the major impediment of flavivirus therapeutic development, is highlighted. These advancements suggest that plant-based systems are excellent alternatives for addressing the remaining challenges of mAb therapeutic development against flavivirus and may facilitate the eventual commercialization of these drug candidates.

Plant-produced anti-dengue virus monoclonal antibodies exhibit reduced antibody-dependent enhancement of infection activity.

The mAb E60 has the potential to be a desirable therapeutic molecule since it efficiently neutralizes all four serotypes of dengue virus (DENV). However, mammalian-cell-produced E60 exhibits antibody-dependent enhancement of infection (ADE) activity, rendering it inefficacious in vivo, and treated animals more susceptible to developing more severe diseases during secondary infection. In this study, we evaluated a plant-based expression system for the production of therapeutically suitable E60. The mAb was transiently expressed in Nicotiana benthamianaWT and a ∆XFT line, a glycosylation mutant lacking plant-specific N-glycan residues. The mAb was efficiently expressed and assembled in leaves and exhibited highly homogenous N-glycosylation profiles, i.e. GnGnXF3 or GnGn structures, depending on the expression host. Both E60 glycovariants demonstrated equivalent antigen-binding specificity and in vitro neutralization potency against DENV serotypes 2 and 4 compared with their mammalian-cell-produced counterpart. By contrast, plant-produced E60 exhibited reduced ADE activity in Fc gamma receptor expressing human cells. Our results suggest the ability of plant-produced antibodies to minimize ADE, which may lead to the development of safe and highly efficacious antibody-based therapeutics against DENV and other ADE-prone viral diseases. Our study provides so far unknown insight into the relationship between mAb N-glycosylation and ADE, which contributes to our understanding of how sugar moieties of antibodies modulate Fc-mediated functions and viral pathogenesis.

Structural and functional characterization of an anti-West Nile virus monoclonal antibody and its single-chain variant produced in glycoengineered plants.

Previously, our group engineered a plant-derived monoclonal antibody (MAb pE16) that efficiently treated West Nile virus (WNV) infection in mice. In this study, we developed a pE16 variant consisting of a single-chain variable fragment (scFv) fused to the heavy chain constant domains (CH) of human IgG (pE16scFv-CH). pE16 and pE16scFv-CH were expressed and assembled efficiently in Nicotiana benthamiana ∆XF plants, a glycosylation mutant lacking plant-specific N-glycan residues. Glycan analysis revealed that ∆XF plant-derived pE16scFv-CH (∆XFpE16scFv-CH) and pE16 (∆XFpE16) both displayed a mammalian glycosylation profile. ∆XFpE16 and ∆XFpE16scFv-CH demonstrated equivalent antigen-binding affinity and kinetics, and slightly enhanced neutralization of WNV in vitro compared with the parent mammalian cell-produced E16 (mE16). A single dose of ∆XFpE16 or ∆XFpE16scFv-CH protected mice against WNV-induced mortality even 4 days after infection at equivalent rates as mE16. This study provides a detailed tandem comparison of the expression, structure and function of a therapeutic MAb and its single-chain variant produced in glycoengineered plants. Moreover, it demonstrates the development of anti-WNV MAb therapeutic variants that are equivalent in efficacy to pE16, simpler to produce, and likely safer to use as therapeutics due to their mammalian N-glycosylation. This platform may lead to a more robust and cost-effective production of antibody-based therapeutics against WNV infection and other infectious, inflammatory or neoplastic diseases.

Quantitative assessment of multiple pathogen exposure and immune dynamics at scale.

IMPORTANCE: Serology reveals exposure to pathogens, as well as the state of autoimmune and other clinical conditions. It is used to evaluate individuals and their histories and as a public health tool to track epidemics. Employing a variety of formats, studies nearly always perform serology by testing response to only one or a few antigens. However, clinical outcomes of new infections also depend on which previous infections may have occurred. We developed a high-throughput serology method that evaluates responses to hundreds of antigens simultaneously. It can be used to evaluate thousands of samples at a time and provide a quantitative readout. This tool will enable doctors to monitor which pathogens an individual has been exposed to and how that changes in the future. Moreover, public health officials could track populations and look for infectious trends among large populations. Testing many potential antigens at a time may also aid in vaccine development.

Monoclonal antibody produced in plants efficiently treats West Nile virus infection in mice.

Over the past decade, West Nile virus (WNV) has spread to all 48 of the lower United States as well as to parts of Canada, Mexico, the Caribbean, and South America, with outbreaks of neuroinvasive disease occurring annually. At present, no therapeutic or vaccine is available for human use. Epidemics of WNV and other emerging infectious disease threats demand cost-efficient and scalable production technologies that can rapidly transfer effective therapeutics into the clinical setting. We have previously reported that Hu-E16, a humanized anti-WNV mAb, binds to a highly conserved epitope on the envelope protein, blocks viral fusion, and shows promising postexposure therapeutic activity. Herein, we generated a plant-derived Hu-E16 mAb that can be rapidly scaled up for commercial production. Plant Hu-E16 was expressed at high levels within 8 days of infiltration in Nicotiana benthamiana plants and retained high-affinity binding and potent neutralizing activity in vitro against WNV. A single dose of plant Hu-E16 protected mice against WNV-induced mortality even 4 days after infection at rates that were indistinguishable from mammalian-cell-produced Hu-E16. This study demonstrates the efficacy of a plant-produced mAb against a potentially lethal infection several days after exposure in an animal challenge model and provides a proof of principle for the development of plant-derived mAbs as therapy against emerging infectious diseases.

Antibody-Dependent Enhancement Activity of a Plant-Made Vaccine against West Nile Virus.

West Nile virus (WNV) causes annual outbreaks globally and is the leading cause of mosquito-borne disease in Unite States. In the absence of licensed therapeutics, there is an urgent need to develop effective and safe human vaccines against WNV. One of the major safety concerns for WNV vaccine development is the risk of increasing infection by related flaviviruses in vaccinated subjects via antibody-dependent enhancement of infection (ADE). Herein, we report the development of a plant-based vaccine candidate that provides protective immunity against a lethal WNV challenge mice, while minimizes the risk of ADE for infection by Zika (ZIKV) and dengue (DENV) virus. Specifically, a plant-produced virus-like particle (VLP) that displays the WNV Envelope protein domain III (wDIII) elicited both high neutralizing antibody titers and antigen-specific cellular immune responses in mice. Passive transfer of serum from VLP-vaccinated mice protected recipient mice from a lethal challenge of WNV infection. Notably, VLP-induced antibodies did not enhance the infection of Fc gamma receptor-expressing K562 cells by ZIKV or DENV through ADE. Thus, a plant-made wDIII-displaying VLP presents a promising WNV vaccine candidate that induces protective immunity and minimizes the concern of inducing ADE-prone antibodies to predispose vaccinees to severe infection by DENV or ZIKV.

Plant-Produced Anti-Zika Virus Monoclonal Antibody Glycovariant Exhibits Abrogated Antibody-Dependent Enhancement of Infection.

Monoclonal antibodies (mAb) against the envelope (E) protein of Zika virus (ZIKV) have shown great potential as therapeutics against the Zika epidemics. However, their use as a therapy may predispose treated individuals to severe infection by the related dengue virus (DENV) via antibody-dependent enhancement of infection (ADE). Here, we generated a broadly neutralizing flavivirus mAb, ZV1, with an identical protein backbone but different Fc glycosylation profiles. The three glycovariants, produced in wild-type (WT) and glycoengineered ΔXF Nicotiana benthamiana plants and in Chinese hamster ovary cells (ZV1WT, ZV1ΔXF, and ZV1CHO), respectively, showed equivalent neutralization potency against both ZIKV and DENV. By contrast, the three mAb glycoforms demonstrated drastically different ADE activity for DENV and ZIKV infection. While ZV1CHO and ZV1ΔXF showed ADE activity upon DENV and ZIKV infection, ZV1WT totally forwent its ADE. Importantly, all three glycovariants exhibited antibody-dependent cellular cytotoxicity (ADCC) against virus-infected cells, with increased potency by the fucose-free ZV1ΔXF glycoform. Moreover, the in vivo efficacy of the ADE-free ZV1WT was demonstrated in a murine model. Collectively, we demonstrated the feasibility of modulating ADE by Fc glycosylation, thereby establishing a novel approach for improving the safety of flavivirus therapeutics. Our study also underscores the versatile use of plants for the rapid expression of complex human proteins to reveal novel insight into antibody function and viral pathogenesis.

A Monoclonal Antibody Produced in Glycoengineered Plants Potently Neutralizes Monkeypox Virus.

The 2022 global outbreaks of monkeypox virus (MPXV) and increased human-to-human transmission calls for the urgent development of countermeasures to protect people who cannot benefit from vaccination. Here, we describe the development of glycovariants of 7D11, a neutralizing monoclonal IgG antibody (mAb) directed against the L1 transmembrane protein of the related vaccinia virus, in a plant-based system as a potential therapeutic against the current MPVX outbreak. Our results indicated that 7D11 mAb quickly accumulates to high levels within a week after gene introduction to plants. Plant-produced 7D11 mAb assembled correctly into the tetrameric IgG structure and can be easily purified to homogeneity. 7D11 mAb exhibited a largely homogeneous N-glycosylation profile, with or without plant-specific xylose and fucose residues, depending on the expression host, namely wild-type or glycoengineered plants. Plant-made 7D11 retained specific binding to its antigen and displayed a strong neutralization activity against MPXV, as least as potent as the reported activity against vaccinia virus. Our study highlights the utility of anti-L1 mAbs as MPXV therapeutics, and the use of glycoengineered plants to develop mAb glycovariants for potentially enhancing the efficacy of mAbs to combat ever-emerging/re-emerging viral diseases.

A Dual-Approach Strategy to Optimize the Safety and Efficacy of Anti-Zika Virus Monoclonal Antibody Therapeutics.

Antibody-dependent enhancement of infection (ADE) is clinically relevant to Dengue virus (DENV) infection and poses a major risk to the application of monoclonal antibody (mAb)-based therapeutics against related flaviviruses such as the Zika virus (ZIKV). Here, we tested a two-tier approach for selecting non-cross-reactive mAbs combined with modulating Fc glycosylation as a strategy to doubly secure the elimination of ADE while preserving Fc effector functions. To this end, we selected a ZIKV-specific mAb (ZV54) and generated three ZV54 variants using Chinese hamster ovary cells and wild-type (WT) and glycoengineered ΔXF Nicotiana benthamiana plants as production hosts (ZV54CHO, ZV54WT, and ZV54ΔXF). The three ZV54 variants shared an identical polypeptide backbone, but each exhibited a distinct Fc N-glycosylation profile. All three ZV54 variants showed similar neutralization potency against ZIKV but no ADE activity for DENV infection, validating the importance of selecting the virus/serotype-specific mAbs for avoiding ADE by related flaviviruses. For ZIKV infection, however, ZV54CHO and ZV54ΔXF showed significant ADE activity while ZV54WT completely forwent ADE, suggesting that Fc glycan modulation may yield mAb glycoforms that abrogate ADE even for homologous viruses. In contrast to the current strategies for Fc mutations that abrogate all effector functions along with ADE, our approach allowed the preservation of effector functions as all ZV54 glycovariants retained antibody-dependent cellular cytotoxicity (ADCC) against the ZIKV-infected cells. Furthermore, the ADE-free ZV54WT demonstrated in vivo efficacy in a ZIKV-infection mouse model. Collectively, our study provides further support for the hypothesis that antibody-viral surface antigen and Fc-mediated host cell interactions are both prerequisites for ADE, and that a dual-approach strategy, as shown herein, contributes to the development of highly safe and efficacious anti-ZIKV mAb therapeutics. Our findings may be impactful to other ADE-prone viruses, including SARS-CoV-2.

Robust production of virus-like particles and monoclonal antibodies with geminiviral replicon vectors in lettuce.

Pharmaceutical protein production in plants has been greatly promoted by the development of viral-based vectors and transient expression systems. Tobacco and related Nicotiana species are currently the most common host plants for the generation of plant-made pharmaceutical proteins (PMPs). Downstream processing of target PMPs from these plants, however, is hindered by potential technical and regulatory difficulties owing to the presence of high levels of phenolics and toxic alkaloids. Here, we explored the use of lettuce, which grows quickly yet produces low levels of secondary metabolites and viral vector-based transient expression systems to develop a robust PMP production platform. Our results showed that a geminiviral replicon system based on the bean yellow dwarf virus permits high-level expression in lettuce of virus-like particles (VLP) derived from the Norwalk virus capsid protein and therapeutic monoclonal antibodies (mAbs) against Ebola and West Nile viruses. These vaccine and therapeutic candidates can be readily purified from lettuce leaves with scalable processing methods while fully retaining functional activity. Furthermore, this study also demonstrated the feasibility of using commercially produced lettuce for high-level PMP production. This allows our production system to have access to unlimited quantities of inexpensive plant material for large-scale production. These results establish a new production platform for biological pharmaceutical agents that are effective, safe, low cost, and amenable to large-scale manufacturing.

A novel system for rapid and cost-effective production of detection and diagnostic reagents of West Nile virus in plants.

The threat of West Nile virus (WNV) epidemics necessitates the development of a technology platform that can produce reagents to support detection and diagnosis rapidly and inexpensively. A plant expression system is attractive for protein production due to its low-cost and high-scalability nature and its ability to make appropriate posttranslational modifications. Here, we investigated the feasibility of using plants to produce two WNV detection and diagnostic reagents to address the current cost and scalability issues. We demonstrated that WNV DIII antigen and E16 monoclonal antibody are rapidly produced at high levels in two plant species and are easily purified. Furthermore, they are effective in identifying WNV and in detecting human IgM response to WNV infection. E16 mAb does not cross-react with other flaviviruses, therefore, is valuable for improving diagnostic accuracy. This study provides a proof of principle for using plants as a robust and economical system to produce diagnostic reagents for arboviruses.

Bioprocessing of plant-derived virus-like particles of Norwalk virus capsid protein under current Good Manufacture Practice regulations.

Despite the success in expressing a variety of subunit vaccine proteins in plants and the recent stride in improving vaccine accumulation levels by transient expression systems, there is still no plant-derived vaccine that has been licensed for human use. The lack of commercial success of plant-made vaccines lies in several technical and regulatory barriers that remain to be overcome. These challenges include the lack of scalable downstream processing procedures, the uncertainty of regulatory compliance of production processes, and the lack of demonstration of plant-derived products that meet the required standards of regulatory agencies in identity, purity, potency and safety. In this study, we addressed these remaining challenges and successfully demonstrate the ability of using plants to produce a pharmaceutical grade Norwalk virus (NV) vaccine under current Good Manufacture Practice (cGMP) guidelines at multiple gram scales. Our results demonstrate that an efficient and scalable extraction and purification scheme can be established for processing virus-like particles (VLPs) of NV capsid protein (NVCP). We successfully operated the upstream and downstream NVCP production processes under cGMP regulations. Furthermore, plant-derived NVCP VLP demonstrates the identity, purity, potency and safety that meet the preset release specifications. This material is being tested in a Phase I human clinical trial. This research provides the first report of producing a plant-derived vaccine at scale under cGMP regulations in an academic setting and an important step for plant-produced vaccines to become a commercial reality.

Potential for a Plant-Made SARS-CoV-2 Neutralizing Monoclonal Antibody as a Synergetic Cocktail Component.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has caused a public health crisis over the last two years. Monoclonal antibody (mAb)-based therapeutics against the spike (S) protein have been shown to be effective treatments for SARS-CoV-2 infection, especially the original viral strain. However, the current mAbs produced in mammalian cells are expensive and might be unaffordable for many. Furthermore, the emergence of variants of concern demands the development of strategies to prevent mutant escape from mAb treatment. Using a cocktail of mAbs that bind to complementary neutralizing epitopes is one such strategy. In this study, we use Nicotiana benthamiana plants in an effort to expedite the development of efficacious and affordable antibody cocktails against SARS-CoV-2. We show that two mAbs can be highly expressed in plants and are correctly assembled into IgG molecules. Moreover, they retain target epitope recognition and, more importantly, neutralize multiple SARS-CoV-2 variants. We also show that one plant-made mAb has neutralizing synergy with other mAbs that we developed in hybridomas. This is the first report of a plant-made mAb to be assessed as a potential component of a SARS-CoV-2 neutralizing cocktail. This work may offer a strategy for using plants to quickly develop mAb cocktail-based therapeutics against emerging viral diseases with high efficacy and low costs.

Expression of an immunogenic Ebola immune complex in Nicotiana benthamiana.

Filoviruses (Ebola and Marburg viruses) cause severe and often fatal haemorrhagic fever in humans and non-human primates. The US Centers for Disease Control identifies Ebola and Marburg viruses as 'category A' pathogens (defined as posing a risk to national security as bioterrorism agents), which has lead to a search for vaccines that could prevent the disease. Because the use of such vaccines would be in the service of public health, the cost of production is an important component of their development. The use of plant biotechnology is one possible way to cost-effectively produce subunit vaccines. In this work, a geminiviral replicon system was used to produce an Ebola immune complex (EIC) in Nicotiana benthamiana. Ebola glycoprotein (GP1) was fused at the C-terminus of the heavy chain of humanized 6D8 IgG monoclonal antibody, which specifically binds to a linear epitope on GP1. Co-expression of the GP1-heavy chain fusion and the 6D8 light chain using a geminiviral vector in leaves of N. benthamiana produced assembled immunoglobulin, which was purified by ammonium sulphate precipitation and protein G affinity chromatography. Immune complex formation was confirmed by assays to show that the recombinant protein bound the complement factor C1q. Size measurements of purified recombinant protein by dynamic light scattering and size-exclusion chromatography also indicated complex formation. Subcutaneous immunization of BALB/C mice with purified EIC resulted in anti-Ebola virus antibody production at levels comparable to those obtained with a GP1 virus-like particle. These results show excellent potential for a plant-expressed EIC as a human vaccine.

Plant-Produced Antigen Displaying Virus-Like Particles Evokes Potent Antibody Responses against West Nile Virus in Mice.

In this study, we developed a hepatitis B core antigen (HBcAg)-based virus-like particle (VLP) that displays the West Nile virus (WNV) Envelope protein domain III (wDIII) as a vaccine candidate for WNV. The HBcAg-wDIII fusion protein was quickly produced in Nicotiana benthamiana plants and reached a high expression level of approximately 1.2 mg of fusion protein per gram of leaf fresh weight within six days post gene infiltration. Electron microscopy and gradient centrifugation analysis indicated that the introduction of wDIII did not interfere with VLP formation and HBcAg-wDIII successfully assembled into VLPs. HBcAg-wDIII VLPs can be easily purified in large quantities from Nicotiana benthamiana leaves to >95% homogeneity. Further analysis revealed that the wDIII was displayed properly and demonstrated specific binding to an anti-wDIII monoclonal antibody that recognizes a conformational epitope of wDIII. Notably, HBcAg-wDIII VLPs were shown to be highly immunogenic and elicited potent humoral responses in mice with antigen-specific IgG titers equivalent to that of protective wDIII antigens in previous studies. Thus, our wDIII-based VLP vaccine offers an attractive option for developing effective, safe, and low-cost vaccines against WNV.

High-level rapid production of full-size monoclonal antibodies in plants by a single-vector DNA replicon system.

Plant viral vectors have great potential in rapid production of important pharmaceutical proteins. However, high-yield production of hetero-oligomeric proteins that require the expression and assembly of two or more protein subunits often suffers problems due to the "competing" nature of viral vectors derived from the same virus. Previously we reported that a bean yellow dwarf virus (BeYDV)-derived, three-component DNA replicon system allows rapid production of single recombinant proteins in plants (Huang et al., 2009. Biotechnol Bioeng 103: 706-714). In this article, we report further development of this expression system for its application in high-yield production of oligomeric protein complexes including monoclonal antibodies (mAbs) in plants. We showed that the BeYDV replicon system permits simultaneous efficient replication of two DNA replicons and thus, high-level accumulation of two recombinant proteins in the same plant cell. We also demonstrated that a single vector that contains multiple replicon cassettes was as efficient as the three-component system in driving the expression of two distinct proteins. Using either the non-competing, three-vector system or the multi-replicon single vector, we produced both the heavy and light chain subunits of a protective IgG mAb 6D8 against Ebola virus GP1 (Wilson et al., 2000. Science 287: 1664-1666) at 0.5 mg of mAb per gram leaf fresh weight within 4 days post-infiltration of Nicotiana benthamiana leaves. We further demonstrated that full-size tetrameric IgG complex containing two heavy and two light chains was efficiently assembled and readily purified, and retained its functionality in specific binding to inactivated Ebola virus. Thus, our single-vector replicon system provides high-yield production capacity for hetero-oligomeric proteins, yet eliminates the difficult task of identifying non-competing virus and the need for co-infection of multiple expression modules. The multi-replicon vector represents a significant advance in transient expression technology for antibody production in plants.

A plant-produced vaccine protects mice against lethal West Nile virus infection without enhancing Zika or dengue virus infectivity.

West Nile virus (WNV) has caused multiple global outbreaks with increased frequency of neuroinvasive disease in recent years. Despite many years of research, there are no licensed therapeutics or vaccines available for human use. One of the major impediments of vaccine development against WNV is the potential enhancement of infection by related flaviviruses in vaccinated subjects through the mechanism of antibody-dependent enhancement of infection (ADE). For instance, the recent finding of enhancement of Zika virus (ZIKV) infection by pre-exposure to WNV further complicates the development of WNV vaccines. Epidemics of WNV and the potential risk of ADE by current vaccine candidates demand the development of effective and safe vaccines. We have previously reported that the domain III (DIII) of the WNV envelope protein can be readily expressed in Nicotiana benthamiana leaves, purified to homogeneity, and promote antigen-specific antibody response in mice. Herein, we further investigated the in vivo potency of a plant-made DIII (plant-DIII) in providing protective immunity against WNV infection. Furthermore, we examined if vaccination with plant-DIII would enhance the risk of a subsequent infection by ZIKV and Dengue virus (DENV). Plant-DIII vaccination evoked antigen-specific cellular immune responses as well as humoral responses. DIII-specific antibodies were neutralizing and the neutralization titers met the threshold correlated with protective immunity by vaccines against multiple flaviviruses. Furthermore, passive administration of anti-plant DIII mouse serum provided full protection against a lethal challenge of WNV infection in mice. Notably, plant DIII-induced antibodies did not enhance ZIKV and DENV infection in Fc gamma receptor-expressing cells, addressing the concern of WNV vaccines in inducing cross-reactive antibodies and sensitizing subjects to subsequent infection by heterologous flavivirus. This study provides the first report of a WNV subunit vaccine that induces protective immunity, while circumventing induction of antibodies with enhancing activity for ZIKV and DENV infection.