Heterologous prime-boost vaccination drives early maturation of HIV broadly neutralizing antibody precursors in humanized mice.

A protective HIV vaccine will likely need to induce broadly neutralizing antibodies (bnAbs). Vaccination with the germline-targeting immunogen eOD-GT8 60mer adjuvanted with AS01B was found to induce VRC01-class bnAb precursors in 97% of vaccine recipients in the IAVI G001 phase 1 clinical trial; however, heterologous boost immunizations with antigens more similar to the native glycoprotein will be required to induce bnAbs. Therefore, we designed core-g28v2 60mer, a nanoparticle immunogen to be used as a first boost after eOD-GT8 60mer priming. We found, using a humanized mouse model approximating human conditions of VRC01-class precursor B cell diversity, affinity, and frequency, that both protein- and mRNA-based heterologous prime-boost regimens induced VRC01-class antibodies that gained key mutations and bound to near-native HIV envelope trimers lacking the N276 glycan. We further showed that VRC01-class antibodies induced by mRNA-based regimens could neutralize pseudoviruses lacking the N276 glycan. These results demonstrated that heterologous boosting can drive maturation toward VRC01-class bnAb development and supported the initiation of the IAVI G002 phase 1 trial testing mRNA-encoded nanoparticle prime-boost regimens.

Structural conservation of Lassa virus glycoproteins and recognition by neutralizing antibodies.

Lassa fever is an acute hemorrhagic fever caused by the zoonotic Lassa virus (LASV). The LASV glycoprotein complex (GPC) mediates viral entry and is the sole target for neutralizing antibodies. Immunogen design is complicated by the metastable nature of recombinant GPCs and the antigenic differences among phylogenetically distinct LASV lineages. Despite the sequence diversity of the GPC, structures of most lineages are lacking. We present the development and characterization of prefusion-stabilized, trimeric GPCs of LASV lineages II, V, and VII, revealing structural conservation despite sequence diversity. High-resolution structures and biophysical characterization of the GPC in complex with GP1-A-specific antibodies suggest their neutralization mechanisms. Finally, we present the isolation and characterization of a trimer-preferring neutralizing antibody belonging to the GPC-B competition group with an epitope that spans adjacent protomers and includes the fusion peptide. Our work provides molecular detail information on LASV antigenic diversity and will guide efforts to design pan-LASV vaccines.

Deep repertoire mining uncovers ultra-broad coronavirus neutralizing antibodies targeting multiple spike epitopes.

Development of vaccines and therapeutics that are broadly effective against known and emergent coronaviruses is an urgent priority. Current strategies for developing pan-coronavirus countermeasures have largely focused on the receptor binding domain (RBD) and S2 regions of the coronavirus Spike protein; it has been unclear whether the N-terminal domain (NTD) is a viable target for universal vaccines and broadly neutralizing antibodies (Abs). Additionally, many RBD-targeting Abs have proven susceptible to viral escape. We screened the circulating B cell repertoires of COVID-19 survivors and vaccinees using multiplexed panels of uniquely barcoded antigens in a high-throughput single cell workflow to isolate over 9,000 SARS-CoV-2-specific monoclonal Abs (mAbs), providing an expansive view of the SARS-CoV-2-specific Ab repertoire. We observed many instances of clonal coalescence between individuals, suggesting that Ab responses frequently converge independently on similar genetic solutions. Among the recovered antibodies was TXG-0078, a public neutralizing mAb that binds the NTD supersite region of the coronavirus Spike protein and recognizes a diverse collection of alpha- and beta-coronaviruses. TXG-0078 achieves its exceptional binding breadth while utilizing the same VH1-24 variable gene signature and heavy chain-dominant binding pattern seen in other NTD supersite-specific neutralizing Abs with much narrower specificity. We also report the discovery of CC24.2, a pan-sarbecovirus neutralizing mAb that targets a novel RBD epitope and shows similar neutralization potency against all tested SARS-CoV-2 variants, including BQ.1.1 and XBB.1.5. A cocktail of TXG-0078 and CC24.2 provides protection against in vivo challenge with SARS-CoV-2, suggesting potential future use in variant-resistant therapeutic Ab cocktails and as templates for pan-coronavirus vaccine design.

Efficient isolation of rare B cells using next-generation antigen barcoding.

The ability to efficiently isolate antigen-specific B cells in high throughput will greatly accelerate the discovery of therapeutic monoclonal antibodies (mAbs) and catalyze rational vaccine development. Traditional mAb discovery is a costly and labor-intensive process, although recent advances in single-cell genomics using emulsion microfluidics allow simultaneous processing of thousands of individual cells. Here we present a streamlined method for isolation and analysis of large numbers of antigen-specific B cells, including next generation antigen barcoding and an integrated computational framework for B cell multi-omics. We demonstrate the power of this approach by recovering thousands of antigen-specific mAbs, including the efficient isolation of extremely rare precursors of VRC01-class and IOMA-class broadly neutralizing HIV mAbs.

Impact of fecal microbiota transplantation with capsules on the prevention of metabolic syndrome among patients with obesity.

BACKGROUND: Fecal microbiota transplantation (FMT) has been studied for the treatment of metabolic syndrome with varying success. However, the possibility of utilizing FMT to prevent metabolic syndrome is to date unknown. METHODS: Secondary analysis of a previously published double-blind, randomized, placebo-controlled pilot trial of FMT in obese metabolically healthy patients was conducted. Post-prandial glucose and insulin levels were measured (NCT02741518). RESULTS: A total of 22 patients were enrolled, 11 in each arm. There were no baseline differences in the area under the curve (AUC) of glucose or insulin in the FMT group compared to placebo. There was a significant change in glucose AUC at week 12 compared to baseline, and in the insulin AUC at week 6 compared to baseline in the FMT group vs. placebo (change in glucose AUC (mg/dl × 60 min): 579 vs 1978, p = 0.03) (change in insulin AUC (µU/ml × 60 min): 137 vs 2728, p = 0.01). CONCLUSIONS: These data suggest that FMT may have a potential role in preventing the development of metabolic syndrome in patients with obesity.

Inflammatory Bowel Disease Outcomes Following Fecal Microbiota Transplantation for Recurrent C. difficile Infection.

BACKGROUND: Recurrent Clostridioides difficile infection (CDI) in patients with inflammatory bowel disease (IBD) is a clinical challenge. Fecal microbiota transplantation (FMT) has emerged as a recurrent CDI therapy. Anecdotal concerns exist regarding worsening of IBD activity; however, prospective data among IBD patients are limited. METHODS: Secondary analysis from an open-label, prospective, multicenter cohort study among IBD patients with 2 or more CDI episodes was performed. Participants underwent a single FMT by colonoscopy (250 mL, healthy universal donor). Secondary IBD-related outcomes included rate of de novo IBD flares, worsening IBD, and IBD improvement-all based on Mayo or Harvey-Bradshaw index (HBI) scores. Stool samples were collected for microbiome and targeted metabolomic profiling. RESULTS: Fifty patients enrolled in the study, among which 15 had Crohn's disease (mean HBI, 5.8 ± 3.4) and 35 had ulcerative colitis (mean partial Mayo score, 4.2 ± 2.1). Overall, 49 patients received treatment. Among the Crohn's disease cohort, 73.3% (11 of 15) had IBD improvement, and 4 (26.6%) had no disease activity change. Among the ulcerative colitis cohort, 62% (22 of 34) had IBD improvement, 29.4% (11 of 34) had no change, and 4% (1 of 34) experienced a de novo flare. Alpha diversity significantly increased post-FMT, and ulcerative colitis patients became more similar to the donor than Crohn's disease patients (P = 0.04). CONCLUSION: This prospective trial assessing FMT in IBD-CDI patients suggests IBD outcomes are better than reported in retrospective studies.

Rapid isolation of potent SARS-CoV-2 neutralizing antibodies and protection in a small animal model.

The development of countermeasures to prevent and treat COVID-19 is a global health priority. In under 7 weeks, we enrolled a cohort of SARS-CoV-2-recovered participants, developed neutralization assays to interrogate serum and monoclonal antibody responses, adapted our high throughput antibody isolation, production and characterization pipeline to rapidly screen over 1000 antigen-specific antibodies, and established an animal model to test protection. We report multiple highly potent neutralizing antibodies (nAbs) and show that passive transfer of a nAb provides protection against high-dose SARS-CoV-2 challenge in Syrian hamsters. The study suggests a role for nAbs in prophylaxis, and potentially therapy, of COVID-19. The nAbs define protective epitopes to guide vaccine design.

Isolation of potent SARS-CoV-2 neutralizing antibodies and protection from disease in a small animal model.

Countermeasures to prevent and treat coronavirus disease 2019 (COVID-19) are a global health priority. We enrolled a cohort of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-recovered participants, developed neutralization assays to investigate antibody responses, adapted our high-throughput antibody generation pipeline to rapidly screen more than 1800 antibodies, and established an animal model to test protection. We isolated potent neutralizing antibodies (nAbs) to two epitopes on the receptor binding domain (RBD) and to distinct non-RBD epitopes on the spike (S) protein. As indicated by maintained weight and low lung viral titers in treated animals, the passive transfer of a nAb provides protection against disease in high-dose SARS-CoV-2 challenge in Syrian hamsters. The study suggests a role for nAbs in prophylaxis, and potentially therapy, of COVID-19. The nAbs also define protective epitopes to guide vaccine design.

Effects of Fecal Microbiota Transplantation With Oral Capsules in Obese Patients.

BACKGROUND & AIMS: Studies in mice have shown that the intestinal microbiota can contribute to obesity via the anorexigenic gut hormone glucagon-like peptide 1 (GLP1) and bile acids, which affect lipid metabolism. We performed a randomized, placebo-controlled, pilot study of the effects of fecal microbiota transplantation (FMT) in obese, metabolically uncompromised patients. METHODS: We performed a double-blind study of 22 obese patients (body mass index [BMI] ≥5 kg/m2) without a diagnosis of diabetes, nonalcoholic steatohepatitis, or metabolic syndrome. Participants were assigned randomly (1:1) to groups that received FMT by capsules (induction dose of 30 capsules at week 4 and maintenance dose of 12 capsules at week 8) or placebo capsules. FMT capsules were derived from a single lean donor (BMI, 17.5 kg/m2). Patients were followed up through week 26; the primary outcome was safety. Stool and serum samples were collected from patients at baseline and at weeks 1, 4, 6, 8, and 12 after administration of the first dose of FMT or placebo and analyzed by 16S RNA gene sequencing. Stool and serum samples were analyzed for metabolomics by liquid chromatography-mass spectrometry. Additional outcomes were the change in area under the curve for GLP1 at week 12. RESULTS: We observed no significant differences in adverse events between patients who received FMT vs placebo. There was no increase in the area under the curve of GLP1 in either group. Patients who received FMT had sustained shifts in microbiomes associated with obesity toward those of the donor (P < .001). Patients who received FMT had a sustained decrease in stool levels of taurocholic acid (P < .05) compared with baseline; bile acid profiles began to resemble those of the donor more closely. We did not observe significant changes in mean BMI at week 12 in either group. CONCLUSIONS: In a placebo-controlled pilot study, we found that FMT capsules (derived from a lean donor) were safe but did not reduce BMI in obese metabolically uncompromised patients. The FMT capsules were well tolerated and led to sustained changes in the intestinal microbiome and bile acid profiles that were similar to those of the lean donor. ClinicalTrials.gov number: NCT02741518.

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.

PTEN Physically Interacts with and Regulates E2F1-mediated Transcription in Lung Cancer.

PTEN phosphorylation at its C-terminal (C-tail) serine/threonine cluster negatively regulates its tumor suppressor function. However, the consequence of such inhibition and its downstream effects in driving lung cancer remain unexplored. Herein, we ascertain the molecular mechanisms by which phosphorylation compromises PTEN function, contributing to lung cancer. Replacement of the serine/threonine residues with alanine generated PTEN-4A, a phosphorylation-deficient PTEN mutant, which suppressed lung cancer cell proliferation and migration. PTEN-4A preferentially localized to the nucleus where it suppressed E2F1-mediated transcription of cell cycle genes. PTEN-4A physically interacted with the transcription factor E2F1 and associated with chromatin at gene promoters with E2F1 DNA-binding sites, a likely mechanism for its transcriptional suppression function. Deletion analysis revealed that the C2 domain of PTEN was indispensable for suppression of E2F1-mediated transcription. Further, we uncovered cancer-associated C2 domain mutant proteins that had lost their ability to suppress E2F1-mediated transcription, supporting the concept that these mutations are oncogenic in patients. Consistent with these findings, we observed increased PTEN phosphorylation and reduced nuclear PTEN levels in lung cancer patient samples establishing phosphorylation as a bona fide inactivation mechanism for PTEN in lung cancer. Thus, use of small molecule inhibitors that hinder PTEN phosphorylation is a plausible approach to activate PTEN function in the treatment of lung cancer. Abbreviations AKT V-Akt Murine Thymoma Viral Oncogene CA Cancer adjacent CDK1 Cyclin dependent kinase 1 CENPC-C Centromere Protein C ChIP Chromatin Immunoprecipitation co-IP Co-immunoprecipitation COSMIC Catalog of Somatic Mutations In Cancer CREB cAMP Responsive Element Binding Protein C-tail Carboxy terminal tail E2F1 E2F Transcription Factor 1 ECIS Electric Cell-substrate Impedance Sensing EGFR Epidermal Growth Factor Receptor GSI Gamma Secretase Inhibitor HDAC1 Histone Deacetylase 1 HP1 Heterochromatin protein 1 KAP1/TRIM28 KRAB-Associated Protein 1/Tripartite Motif Containing 28 MAF1 Repressor of RNA polymerase III transcription MAF1 homolog MCM2 Minichromosome Maintenance Complex Component 2 miRNA micro RNA MTF1 Metal-Regulatory Transcription Factor 1 PARP Poly(ADP-Ribose) Polymerase PD-1 Programmed Cell Death 1 PD-L1 Programmed Cell Death 1 Ligand 1 PI3K Phosphatidylinositol-4,5-Bisphosphate 3-Kinase PLK Polo-like Kinase pPTEN Phosphorylated PTEN PTEN Phosphatase and Tensin Homolog deleted on chromosome ten PTM Post Translational Modification Rad51 RAD51 Recombinase Rad52 RAD52 Recombinase RPA1 Replication protein A SILAC Stable Isotope Labeling with Amino Acids in Cell Culture SRF Serum Response Factor TKI Tyrosine Kinase inhbitors TMA Tissue Microarray TOP2A DNA Topoisomerase 2A.

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.

Transient Protein Expression by Agroinfiltration in Lettuce.

Current systems of recombinant protein production include bacterial, insect, and mammalian cell culture. However, these platforms are expensive to build and operate at commercial scales and/or have limited abilities to produce complex proteins. In recent years, plant-based expression systems have become top candidates for the production of recombinant proteins as they are highly scalable, robust, safe, and can produce complex proteins due to having a eukaryotic endomembrane system. Newly developed "deconstructed" viral vectors delivered via Agrobacterium tumefaciens (agroinfiltration) have enabled robust plant-based production of proteins with a wide range of applications. The leafy Lactuca sativa (lettuce) plant with its strong foundation in agriculture is an excellent host for pharmaceutical protein production. Here, we describe a method for agroinfiltration of lettuce that can rapidly produce high levels of recombinant proteins in a matter of days and has the potential to be scaled up to an agricultural level.

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.

A plant-produced antigen elicits potent immune responses against West Nile virus in mice.

We described the rapid production of the domain III (DIII) of the envelope (E) protein in plants as a vaccine candidate for West Nile Virus (WNV). Using various combinations of vector modules of a deconstructed viral vector expression system, DIII was produced in three subcellular compartments in leaves of Nicotiana benthamiana by transient expression. DIII expressed at much higher levels when targeted to the endoplasmic reticulum (ER) than that targeted to the chloroplast or the cytosol, with accumulation level up to 73 µ g DIII per gram of leaf fresh weight within 4 days after infiltration. Plant ER-derived DIII was soluble and readily purified to > 95% homogeneity without the time-consuming process of denaturing and refolding. Further analysis revealed that plant-produced DIII was processed properly and demonstrated specific binding to an anti-DIII monoclonal antibody that recognizes a conformational epitope. Furthermore, subcutaneous immunization of mice with 5 and 25 µ g of purified DIII elicited a potent systemic response. This study provided the proof of principle for rapidly producing immunogenic vaccine candidates against WNV in plants with low cost and scalability.

Efficient agroinfiltration of plants for high-level transient expression of recombinant proteins.

Mammalian cell culture is the major platform for commercial production of human vaccines and therapeutic proteins. However, it cannot meet the increasing worldwide demand for pharmaceuticals due to its limited scalability and high cost. Plants have shown to be one of the most promising alternative pharmaceutical production platforms that are robust, scalable, low-cost and safe. The recent development of virus-based vectors has allowed rapid and high-level transient expression of recombinant proteins in plants. To further optimize the utility of the transient expression system, we demonstrate a simple, efficient and scalable methodology to introduce target-gene containing Agrobacterium into plant tissue in this study. Our results indicate that agroinfiltration with both syringe and vacuum methods have resulted in the efficient introduction of Agrobacterium into leaves and robust production of two fluorescent proteins; GFP and DsRed. Furthermore, we demonstrate the unique advantages offered by both methods. Syringe infiltration is simple and does not need expensive equipment. It also allows the flexibility to either infiltrate the entire leave with one target gene, or to introduce genes of multiple targets on one leaf. Thus, it can be used for laboratory scale expression of recombinant proteins as well as for comparing different proteins or vectors for yield or expression kinetics. The simplicity of syringe infiltration also suggests its utility in high school and college education for the subject of biotechnology. In contrast, vacuum infiltration is more robust and can be scaled-up for commercial manufacture of pharmaceutical proteins. It also offers the advantage of being able to agroinfiltrate plant species that are not amenable for syringe infiltration such as lettuce and Arabidopsis. Overall, the combination of syringe and vacuum agroinfiltration provides researchers and educators a simple, efficient, and robust methodology for transient protein expression. It will greatly facilitate the development of pharmaceutical proteins and promote science education.

Agroinfiltration as an Effective and Scalable Strategy of Gene Delivery for Production of Pharmaceutical Proteins.

Current human biologics are most commonly produced by mammalian cell culture-based fermentation technologies. However, its limited scalability and high cost prevent this platform from meeting the ever increasing global demand. Plants offer a novel alternative system for the production of pharmaceutical proteins that is more scalable, cost-effective, and safer than current expression paradigms. The recent development of deconstructed virus-based vectors has allowed rapid and high-level transient expression of recombinant proteins, and in turn, provided a preferred plant based production platform. One of the remaining challenges for the commercial application of this platform was the lack of a scalable technology to deliver the transgene into plant cells. Therefore, this review focuses on the development of an effective and scalable technology for gene delivery in plants. Direct and indirect gene delivery strategies for plant cells are first presented, and the two major gene delivery technologies based on agroinfiltration are subsequently discussed. Furthermore, the advantages of syringe and vacuum infiltration as gene delivery methodologies are extensively discussed, in context of their applications and scalability for commercial production of human pharmaceutical proteins in plants. The important steps and critical parameters for the successful implementation of these strategies are also detailed in the review. Overall, agroinfiltration based on syringe and vacuum infiltration provides an efficient, robust and scalable gene-delivery technology for the transient expression of recombinant proteins in plants. The development of this technology will greatly facilitate the realization of plant transient expression systems as a premier platform for commercial production of pharmaceutical proteins.