Implications of O-glycan modifications in the hinge region of a plant-produced SARS-CoV-2-IgA antibody on functionality.

Introduction: Prolyl-4-hydroxylases (P4H) catalyse the irreversible conversion of proline to hydroxyproline, constituting a common posttranslational modification of proteins found in humans, plants, and microbes. Hydroxyproline residues can be further modified in plants to yield glycoproteins containing characteristic O-glycans. It is currently unknown how these plant endogenous modifications impact protein functionality and they cause considerable concerns for the recombinant production of therapeutic proteins in plants. In this study, we carried out host engineering to generate a therapeutic glycoprotein largely devoid of plant-endogenous O-glycans for functional characterization. Methods: Genome editing was used to inactivate two genes coding for enzymes of the P4H10 subfamily in the widely used expression host Nicotiana benthamiana. Using glycoengineering in plants and expression in human HEK293 cells we generated four variants of a potent, SARS-CoV-2 neutralizing antibody, COVA2-15 IgA1. The variants that differed in the number of modified proline residues and O-glycan compositions of their hinge region were assessed regarding their physicochemical properties and functionality. Results: We found that plant endogenous O-glycan formation was strongly reduced on IgA1 when transiently expressed in the P4H10 double mutant N. benthamiana plant line. The IgA1 glycoforms displayed differences in proteolytic stability and minor differences in receptor binding thus highlighting the importance of O-glycosylation in the hinge region of human IgA1. Discussion: This work reports the successful protein O-glycan engineering of an important plant host for recombinant protein expression. While the complete removal of endogenous hydroxyproline residues from the hinge region of plant-produced IgA1 is yet to be achieved, our engineered line is suitable for structure-function studies of O-glycosylated recombinant glycoproteins produced in plants.

Marked enhancement of the immunogenicity of plant-expressed IgG-Fc fusion proteins by inclusion of cholera toxin non-toxic B subunit within the single polypeptide.

Immunoglobulin G (IgG)-based fusion proteins have been widely exploited as a potential vaccine delivery platform but in the absence of exogenous adjuvants, the lack of robust immunity remains an obstacle. Here, we report on a key modification that overcomes that obstacle. Thus, we constructed an IgG-Fc vaccine platform for dengue, termed D-PCF, which in addition to a dengue antigen incorporates the cholera toxin non-toxic B subunit (CTB) as a molecular adjuvant, with all three proteins expressed as a single polypeptide. Following expression in Nicotiana benthamiana plants, the D-PCF assembled as polymeric structures of similar size to human IgM, a process driven by the pentamerization of CTB. A marked improvement of functional properties in vitro and immunogenicity in vivo over a previous iteration of the Fc-fusion protein without CTB [1] was demonstrated. These include enhanced antigen presenting cell binding, internalization and activation, complement activation, epithelial cell interactions and ganglioside binding, as well as more efficient polymerization within the expression host. Following immunization of mice with D-PCF by a combination of systemic and mucosal (intranasal) routes, we observed robust systemic and mucosal immune responses, as well as systemic T cell responses, significantly higher than those induced by a related Fc-fusion protein but without CTB. The induced antibodies could bind to the domain III of the dengue virus envelope protein from all four dengue serotypes. Finally, we also demonstrated feasibility of aerosolization of D-PCF as a prerequisite for vaccine delivery by the respiratory route.

Recombinant neutralizing secretory IgA antibodies for preventing mucosal acquisition and transmission of SARS-CoV-2.

Passive delivery of antibodies to mucosal sites may be a valuable adjunct to COVID-19 vaccination to prevent infection, treat viral carriage, or block transmission. Neutralizing monoclonal IgG antibodies are already approved for systemic delivery, and several clinical trials have been reported for delivery to mucosal sites where SARS-CoV-2 resides and replicates in early infection. However, secretory IgA may be preferred because the polymeric complex is adapted for the harsh, unstable external mucosal environment. Here, we investigated the feasibility of producing neutralizing monoclonal IgA antibodies against SARS-CoV-2. We engineered two class-switched mAbs that express well as monomeric and secretory IgA (SIgA) variants with high antigen-binding affinities and increased stability in mucosal secretions compared to their IgG counterparts. SIgAs had stronger virus neutralization activities than IgG mAbs and were protective against SARS-CoV-2 infection in an in vivo murine model. Furthermore, SIgA1 can be aerosolized for topical delivery using a mesh nebulizer. Our findings provide a persuasive case for developing recombinant SIgAs for mucosal application as a new tool in the fight against COVID-19.

Characterisation of two snake toxin-targeting human monoclonal immunoglobulin G antibodies expressed in tobacco plants.

Current snakebite antivenoms are based on polyclonal animal-derived antibodies, which can neutralize snake venom toxins in envenomed victims, but which are also associated with adverse reactions. Therefore, several efforts within antivenom research aim to explore the utility of recombinant monoclonal antibodies, such as human immunoglobulin G (IgG) antibodies, which are routinely used in the clinic for other indications. In this study, the feasibility of using tobacco plants as bioreactors for expressing full-length human monoclonal IgG antibodies against snake toxins was investigated. We show that the plant-produced antibodies perform similarly to their mammalian cell-expressed equivalents in terms of in vitro antigen binding. Complete neutralization was achieved by both the plant and mammalian cell-produced anti-α-cobratoxin antibody. The feasibility of using plant-based expression systems may potentially make it easier for laboratories in resource-poor settings to work with human monoclonal IgG antibodies.

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.

Improving the efficacy of plant-made anti-HIV monoclonal antibodies for clinical use.

Introduction: Broadly neutralising antibodies are promising candidates for preventing and treating Human Immunodeficiency Virus/Acquired Immunodeficiency Syndrome (HIV/AIDS), as an alternative to or in combination with antiretroviral therapy (ART). These mAbs bind to sites on the virus essential for virus attachment and entry, thereby inhibiting entry into the host cell. However, the cost and availability of monoclonal antibodies, especially combinations of antibodies, hampers implementation of anti-HIV bNAb therapies in low- to middle- income countries (LMICs) where HIV-1 prevalence is highest. Methods: We have produced three HIV broadly neutralizing antibodies (bNAbs), 10-1074, VRC01 and 3BNC117 in the Nicotiana benthamiana transient expression system. The impact of specific modifications to enhance potency and efficacy were assessed. To prolong half-life and increase bioavailability, a M252Y/S254T/T256E (YTE) or M428L/N434S (LS) mutation was introduced. To increase antibody dependent cellular cytotoxicity (ADCC), we expressed an afucosylated version of each antibody using a glycoengineered plant line. Results: The majority of bNAbs and their variants could be expressed at yields of up to 47 mg/kg. Neither the expression system nor the modifications impacted the neutralization potential of the bNAbs. Afucosylated bNAbs exhibit enhanced ability to bind to FcγRIIIa and trigger ADCC, regardless of the presence of Fc amino acid mutations. Lastly, we demonstrated that Fc-modified variants expressed in plants show enhanced binding to FcRn, which results in a favourable in vivo pharmacokinetic profile compared to their unmodified counterparts. Conclusion: Tobacco plants are suitable expression hosts for anti-HIV bNAbs with increased efficacy and an improved pharmacokinetic profile.

Killer to cure: Expression and production costs calculation of tobacco plant-made cancer-immune checkpoint inhibitors.

Immune checkpoint inhibitors (ICIs) have achieved huge clinical success. However, many still have limited response rates, and are prohibitively costly. There is a need for effective and affordable ICIs, as well as local manufacturing capacity to improve accessibility, especially to low-to-middle income countries (LMICs). Here, we have successfully expressed three key ICIs (anti-PD-1 Nivolumab, anti-NKG2A Monalizumab, and anti-LAG-3 Relatimab) transiently in Nicotiana benthamiana and Nicotiana tabacum plants. The ICIs were expressed with a combination of different Fc regions and glycosylation profiles. They were characterized in terms of protein accumulation levels, target cell binding, binding to human neonatal Fc receptors (hFcRn), human complement component C1q (hC1q) and various Fcγ receptors, as well as protein recovery during purification at 100 mg- and kg-scale. It was found that all ICIs bound to the expected target cells. Furthermore, the recovery during purification, as well as Fcγ receptor binding, can be altered depending on the Fc region used and the glycosylation profiles. This opens the possibility of using these two parameters to fine-tune the ICIs for desired effector functions. A scenario-based production cost model was also generated based on two production scenarios in hypothetical high- and low-income countries. We have shown that the product accumulation and recovery of plant production platforms were as competitive as mammalian cell-based platforms. This highlights the potential of plants to deliver ICIs that are more affordable and accessible to a widespread market, including LMICs.

Engineering the N-glycosylation pathway of Nicotiana tabacum for molecular pharming using CRISPR/Cas9.

Molecular pharming in plants offers exciting possibilities to address global access to modern biologics. However, differences in the N-glycosylation pathway including the presence of ß(1,2)-xylose and core α(1,3)-fucose can affect activity, potency and immunogenicity of plant-derived proteins. Successful glycoengineering approaches toward human-like structures with no changes in plant phenotype, growth, or recombinant protein expression levels have been reported for Arabidopsis thaliana and Nicotiana benthamiana. Such engineering of N-glycosylation would also be desirable for Nicotiana tabacum, which remains the crop of choice for recombinant protein pharmaceuticals required at massive scale and for manufacturing technology transfer to less developed countries. Here, we generated N. tabacum cv. SR-1 ß(1,2)-xylosyltransferase (XylT) and α(1,3)-fucosyltransferase (FucT) knockout lines using CRISPR/Cas9 multiplex genome editing, targeting three conserved regions of the four FucT and two XylT genes. These two enzymes are responsible for generating non-human N-glycan structures. We confirmed full functional knockout of transformants by immunoblotting of total soluble protein by antibodies recognizing ß(1,2)-xylose and core α(1,3)-fucose, mass spectrometry analysis of recombinantly produced VRC01, a broadly neutralizing anti-HIV-1 hIgG1 antibody, and Sanger sequencing of targeted regions of the putative transformants. These data represent an important step toward establishing Nicotiana tabacum as a biologics platform for Global Health.

Production of Recombinant Proteins in Transgenic Tobacco Plants.

Nicotiana tabacum (the tobacco plant ) has numerous advantages for molecular farming, including rapid growth, large biomass and the possibility of both cross- and self-fertilization. In addition, genetic transformation and tissue culture protocols for regeneration of transgenic plants are well-established. Here, we describe the production of transgenic tobacco using Agrobacterium tumefaciens and the analysis of recombinant proteins, either in crude plant extracts or after purification, by enzyme-linked immunosorbent assays, sodium dodecyl sulfate polyacrylamide gel electrophoresis with western blotting and surface plasmon resonance.

Multiple gene expression in plants using MIDAS-P, a versatile type II restriction-based modular expression vector.

MIDAS-P is a plant expression vector with blue/white screening for iterative cloning of multiple, tandemly arranged transcription units (TUs). We have used the MIDAS-P system to investigate the expression of up to five genes encoding three anti-HIV proteins and the reporter gene DsRed in Nicotiana benthamiana plants. The anti-HIV cocktail was made up of a broadly neutralizing monoclonal antibody (VRC01), a lectin (Griffithsin), and a single-chain camelid nanobody (J3-VHH). Constructs containing different combinations of 3, 4, or 5 TUs encoding different components of the anti-HIV cocktail were assembled. Messenger RNA (mRNA) levels of the genes of interest decreased beyond two TUs. Coexpression of the RNA silencing suppressor P19 dramatically increased the overall mRNA and protein expression levels of each component. The position of individual TUs in 3 TU constructs did not affect mRNA or protein expression levels. However, their expression dropped to non-detectable levels in constructs with four or more TUs each containing the same promoter and terminator elements, with the exception of DsRed at the first or last position in 5 TU constructs. This drop was alleviated by co-expression of P19. In short, the MIDAS-P system is suitable for the simultaneous expression of multiple proteins in one construct.

Contributions of the international plant science community to the fight against human infectious diseases - part 1: epidemic and pandemic diseases.

Infectious diseases, also known as transmissible or communicable diseases, are caused by pathogens or parasites that spread in communities by direct contact with infected individuals or contaminated materials, through droplets and aerosols, or via vectors such as insects. Such diseases cause ˜17% of all human deaths and their management and control places an immense burden on healthcare systems worldwide. Traditional approaches for the prevention and control of infectious diseases include vaccination programmes, hygiene measures and drugs that suppress the pathogen, treat the disease symptoms or attenuate aggressive reactions of the host immune system. The provision of vaccines and biologic drugs such as antibodies is hampered by the high cost and limited scalability of traditional manufacturing platforms based on microbial and animal cells, particularly in developing countries where infectious diseases are prevalent and poorly controlled. Molecular farming, which uses plants for protein expression, is a promising strategy to address the drawbacks of current manufacturing platforms. In this review article, we consider the potential of molecular farming to address healthcare demands for the most prevalent and important epidemic and pandemic diseases, focussing on recent outbreaks of high-mortality coronavirus infections and diseases that disproportionately affect the developing world.

Characterisation of a highly potent and near pan-neutralising anti-HIV monoclonal antibody expressed in tobacco plants.

BACKGROUND: HIV remains one of the most important health issues worldwide, with almost 40 million people living with HIV. Although patients develop antibodies against the virus, its high mutation rate allows evasion of immune responses. Some patients, however, produce antibodies that are able to bind to, and neutralise different strains of HIV. One such 'broadly neutralising' antibody is 'N6'. Identified in 2016, N6 can neutralise 98% of HIV-1 isolates with a median IC50 of 0.066 µg/mL. This neutralisation breadth makes N6 a very promising therapeutic candidate. RESULTS: N6 was expressed in a glycoengineered line of N. benthamiana plants (pN6) and compared to the mammalian cell-expressed equivalent (mN6). Expression at 49 mg/kg (fresh leaf tissue) was achieved in plants, although extraction and purification are more challenging than for most plant-expressed antibodies. N-glycoanalysis demonstrated the absence of xylosylation and a reduction in α(1,3)-fucosylation that are typically found in plant glycoproteins. The N6 light chain contains a potential N-glycosylation site, which was modified and displayed more α(1,3)-fucose than the heavy chain. The binding kinetics of pN6 and mN6, measured by surface plasmon resonance, were similar for HIV gp120. pN6 had a tenfold higher affinity for FcγRIIIa, which was reflected in an antibody-dependent cellular cytotoxicity assay, where pN6 induced a more potent response from effector cells than that of mN6. pN6 demonstrated the same potency and breadth of neutralisation as mN6, against a panel of HIV strains. CONCLUSIONS: The successful expression of N6 in tobacco supports the prospect of developing a low-cost, low-tech production platform for a monoclonal antibody cocktail to control HIV in low-to middle income countries.

Investigation of a monoclonal antibody against enterotoxigenic Escherichia coli, expressed as secretory IgA1 and IgA2 in plants.

Passive immunization with antibodies is a promising approach against enterotoxigenic Escherichia coli diarrhea, a prevalent disease in LMICs. The objective of this study was to investigate expression of a monoclonal anti-ETEC CfaE secretory IgA antibody in N. benthamiana plants, with a view to facilitating access to ETEC passive immunotherapy. SIgA1 and SIgA2 forms of mAb 68-81 were produced by co-expressing the light and engineered heavy chains with J chain and secretory component in N. benthamiana. Antibody expression and assembly were compared with CHO-derived antibodies by SDS-PAGE, western blotting, size-exclusion chromatography and LC-MS peptide mapping. N-linked glycosylation was assessed by rapid fluorescence/mass spectrometry and LC-ESI-MS. Susceptibility to gastric digestion was assessed in an in vitro model. Antibody function was compared for antigen binding, a Caco-2 cell-based ETEC adhesion assay, an ETEC hemagglutination inhibition assay and a murine in vivo challenge study. SIgA1 assembly appeared superior to SIgA2 in plants. Both sub-classes exhibited resistance to degradation by simulated gastric fluid, comparable to CHO-produced 68-61 SIgA1. The plant expressed SIgAs had more homogeneous N-glycosylation than CHO-derived SIgAs, but no alteration of in vitro functional activity was observed, including antibodies expressed in a plant line engineered for mammalian-like N glycosylation. The plant-derived SIgA2 mAb demonstrated protection against diarrhea in a murine infection model. Although antibody yield and purification need to be optimized, anti-ETEC SIgA antibodies produced in a low-cost plant platform are functionally equivalent to CHO antibodies, and provide promise for passive immunotherapy in LMICs.

Rational design and expression of a recombinant plant rhabdovirus glycoprotein for production of immunoreactive murine anti-sera.

Lettuce necrotic yellows virus (LNYV) is a plant rhabdovirus which has a type-1 transmembrane glycoprotein. Here, we describe the generation of murine anti-sera to the glycoprotein. Rational design, expression, and purification of recombinant glycoprotein, termed rLGe, was undertaken using SignalP4.1 and camSol servers to predict signal peptide cleavage and protein solubility. In order to successfully obtain expression in mammalian cells, LNYV glycoprotein native signal peptide was substituted with that of Rabies virus glycoprotein. In addition, rather than expression of the entire molecule, rLGe consisted of the LNYV glycoprotein ectodomain fused to two affinity tags to minimize the risk of protein aggregation, while facilitating detection and purification. rLGe was transiently expressed in mammalian cell culture, purified using affinity column chromatography, and used to immunize mice. Harvested anti-sera were immunoreactive and specific to the naturally occurring glycoprotein as confirmed by western blotting of plant leaf tissue infected with LNYV.

Potential Applications of Plant Biotechnology against SARS-CoV-2.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a novel coronavirus responsible for an ongoing human pandemic (COVID-19). There is a massive international effort underway to develop diagnostic reagents, vaccines, and antiviral drugs in a bid to slow down the spread of the disease and save lives. One part of that international effort involves the research community working with plants, bringing researchers from all over the world together with commercial enterprises to achieve the rapid supply of protein antigens and antibodies for diagnostic kits, and scalable production systems for the emergency manufacturing of vaccines and antiviral drugs. Here, we look at some of the ways in which plants can and are being used in the fight against COVID-19.

Shotguns vs Lasers: Identifying barriers and facilitators to scaling-up plant molecular farming for high-value health products.

Plant molecular farming (PMF) is a convenient and cost-effective way to produce high-value recombinant proteins that can be used in the production of a range of health products, from pharmaceutical therapeutics to cosmetic products. New plant breeding techniques (NPBTs) provide a means to enhance PMF systems more quickly and with greater precision than ever before. However, the feasibility, regulatory standing and social acceptability of both PMF and NPBTs are in question. This paper explores the perceptions of key stakeholders on two European Union (EU) Horizon 2020 programmes-Pharma-Factory and Newcotiana-towards the barriers and facilitators of PMF and NPBTs in Europe. One-on-one qualitative interviews were undertaken with N = 20 individuals involved in one or both of the two projects at 16 institutions in seven countries (Belgium, France, Germany, Italy, Israel, Spain and the UK). The findings indicate that the current EU regulatory environment and the perception of the public towards biotechnology are seen as the main barriers to scaling-up PMF and NPBTs. Competition from existing systems and the lack of plant-specific regulations likewise present challenges for PMF developing beyond its current niche. However, respondents felt that the communication of the benefits and purpose of NPBT PMF could provide a platform for improving the social acceptance of genetic modification. The importance of the media in this process was highlighted. This article also uses the multi-level perspective to explore the ways in which NPBTs are being legitimated by interested parties and the systemic factors that have shaped and are continuing to shape the development of PMF in Europe.

Development of a minigenome cassette for Lettuce necrotic yellows virus: A first step in rescuing a plant cytorhabdovirus.

Rhabdoviruses are enveloped negative-sense RNA viruses that have numerous biotechnological applications. However, recovering plant rhabdoviruses from cDNA remains difficult due to technical difficulties such as the need for concurrent in planta expression of the viral genome together with the viral nucleoprotein (N), phosphoprotein (P) and RNA-dependent RNA polymerase (L) and viral genome instability in E. coli. Here, we developed a negative-sense minigenome cassette for Lettuce necrotic yellows virus (LNYV). We introduced introns into the unstable viral ORF and employed Agrobacterium tumefaciens to co-infiltrate Nicotiana with the genes for the N, P, and L proteins together with the minigenome cassette. The minigenome cassette included the Discosoma sp. red fluorescent protein gene (DsRed) cloned in the negative-sense between the viral trailer and leader sequences which were placed between hammerhead and hepatitis delta ribozymes. In planta DsRed expression was demonstrated by western blotting while the appropriate splicing of introduced introns was confirmed by sequencing of RT-PCR product.

Engineering the interactions between a plant-produced HIV antibody and human Fc receptors.

Plants can provide a cost-effective and scalable technology for production of therapeutic monoclonal antibodies, with the potential for precise engineering of glycosylation. Glycan structures in the antibody Fc region influence binding properties to Fc receptors, which opens opportunities for modulation of antibody effector functions. To test the impact of glycosylation in detail, on binding to human Fc receptors, different glycovariants of VRC01, a broadly neutralizing HIV monoclonal antibody, were generated in Nicotiana benthamiana and characterized. These include glycovariants lacking plant characteristic α1,3-fucose and ß1,2-xylose residues and glycans extended with terminal ß1,4-galactose. Surface plasmon resonance-based assays were established for kinetic/affinity evaluation of antibody-FcγR interactions, and revealed that antibodies with typical plant glycosylation have a limited capacity to engage FcγRI, FcγRIIa, FcγRIIb and FcγRIIIa; however, the binding characteristics can be restored and even improved with targeted glycoengineering. All plant-made glycovariants had a slightly reduced affinity to the neonatal Fc receptor (FcRn) compared with HEK cell-derived antibody. However, this was independent of plant glycosylation, but related to the oxidation status of two methionine residues in the Fc region. This points towards a need for process optimization to control oxidation levels and improve the quality of plant-produced antibodies.

Molecular Pharming for low and middle income countries.

Interest in applications and benefits that Molecular Pharming might offer to Low and Middle Income Countries has always been a potent driver for the research discipline, and a major reason why many scientists entered the field. Although enthusiasm remains high, the reality is that such a game-changing innovation would always take longer than traditional uptake of new technology in developed countries, and be complicated by external factors beyond technical feasibility. Excitingly, signs of increasing interest by LMICS in Molecular Pharming are now emerging. Here, three case studies from Thailand, South Africa and Brazil are used to identify some of the key issues when a new investment into Molecular Pharming manufacturing capacity is under consideration. At present, academic research is not necessarily addressing these issues. Only by understanding the concerns, can members of the academic community contribute to helping the development of Molecular Pharming for LMICs by focusing their research efforts appropriately.

In vitro and in vivo evaluation of a single chain antibody fragment generated in planta with potent rabies neutralisation activity.

Rabies causes more than 60,000 human deaths annually in areas where the virus is endemic. Importantly, rabies is one of the few pathogens for which there is no treatment following the onset of clinical disease with the outcome of infection being death in almost 100% of cases. Whilst vaccination, and the combination of vaccine and rabies immunoglobulin treatment for post-exposure administration are available, no tools have been identified that can reduce or prevent rabies virus replication once clinical disease has initiated. The search for effective antiviral molecules to treat those that have already developed clinical disease associated with rabies virus infection is considered one of the most important goals in rabies research. The current study assesses a single chain antibody molecule (ScFv) based on a monoclonal antibody that potently neutralises rabies in vitro as a potential therapeutic candidate. The recombinant ScFv was generated in Nicotiana benthamiana by transient expression, and was chemically conjugated (ScFv/RVG) to a 29 amino acid peptide, specific for nicotinic acetylcholine receptor (nAchR) binding in the CNS. This conjugated molecule was able to bind nAchR in vitro and enter neuronal cells more efficiently than ScFv. The ability of the ScFv/RVG to neutralise virus in vivo was assessed using a staggered administration where the molecule was inoculated either four hours before, two days after or four days after infection. The ScFv/RVG conjugate was evaluated in direct comparison with HRIG and a potential antiviral molecule, Favipiravir (also known as T-705) to indicate whether there was greater bioavailability of the ScFv in the brains of treated mice. The study indicated that the approach taken with the ScFv/RVG conjugate may have utility in the design and implementation of novel tools targetting rabies virus infection in the brain.