Lack of norovirus replication and histo-blood group antigen expression in 3-dimensional intestinal epithelial cells.

Noroviruses (NoVs) are a leading cause of gastroenteritis worldwide. An in vitro model for NoV replication remains elusive, making study of the virus difficult. A previous study, which used a 3-dimensional (3-D) intestinal model derived from INT-407 cells reported NoV replication and extensive cytopathic effects (CPE). Using the same 3-D model, but with highly purified Norwalk virus (NV), we attempted to replicate this study. Our results showed no evidence of NV replication by real-time PCR of viral RNA or by immunocytochemical detection of viral structural and nonstructural proteins. Immunocytochemical analysis of the 3-D cultures also showed no detectable presence of histo-blood group antigens that participate in NV binding and host tropism. To determine the potential cause of CPE observed in the previous study, we exposed 3-D cultures to lipopolysaccharide concentrations consistent with contaminated stool samples and observed morphologic features similar to CPE. We conclude that the 3-D INT-407 model does not support NV replication.

A nonreplicating subunit vaccine protects mice against lethal Ebola virus challenge.

Ebola hemorrhagic fever is an acute and often deadly disease caused by Ebola virus (EBOV). The possible intentional use of this virus against human populations has led to design of vaccines that could be incorporated into a national stockpile for biological threat reduction. We have evaluated the immunogenicity and efficacy of an EBOV vaccine candidate in which the viral surface glycoprotein is biomanufactured as a fusion to a monoclonal antibody that recognizes an epitope in glycoprotein, resulting in the production of Ebola immune complexes (EICs). Although antigen-antibody immune complexes are known to be efficiently processed and presented to immune effector cells, we found that codelivery of the EIC with Toll-like receptor agonists elicited a more robust antibody response in mice than did EIC alone. Among the compounds tested, polyinosinic:polycytidylic acid (PIC, a Toll-like receptor 3 agonist) was highly effective as an adjuvant agent. After vaccinating mice with EIC plus PIC, 80% of the animals were protected against a lethal challenge with live EBOV (30,000 LD(50) of mouse adapted virus). Surviving animals showed a mixed Th1/Th2 response to the antigen, suggesting this may be important for protection. Survival after vaccination with EIC plus PIC was statistically equivalent to that achieved with an alternative viral vector vaccine candidate reported in the literature. Because nonreplicating subunit vaccines offer the possibility of formulation for cost-effective, long-term storage in biothreat reduction repositories, EIC is an attractive option for public health defense measures.

Intranasal delivery of Norwalk virus-like particles formulated in an in situ gelling, dry powder vaccine.

The development of a vaccine to prevent norovirus infections has been focused on immunization at a mucosal surface, but has been limited by the low immunogenicity of self-assembling Norwalk virus-like particles (NV VLPs) delivered enterically or at nasal surfaces. Nasal immunization, which offers the advantage of ease of immunization, faces obstacles imposed by the normal process of mucociliary clearance, which limits residence time of applied antigens. Herein, we describe the use of a dry powder formulation (GelVac) of an inert in situ gelling polysaccharide (GelSite) extracted from Aloe vera for nasal delivery of NV VLP antigen. Powder formulations, with or without NV VLP antigen, were similar in structure in dry form or when rehydrated in simulated nasal fluids. Immunogenicity of the dry powder VLP formulation was compared to equivalent antigen/adjuvant liquid formulations in animals. For the GelVac powder, we observed superior NV-specific serum and mucosal (aerodigestive and reproductive tracts) antibody responses relative to liquid formulations. Incorporation of the TLR7 agonist gardiquimod in dry powder formulations did not enhance antibody responses, although its inclusion in liquid formulations did enhance VLP immunogenicity irrespective of the presence or absence of GelSite. We interpret these data as showing that GelSite-based dry powder formulations (1) stabilize the immunogenic structural properties of VLPs and (2) induce systemic and mucosal antibody titers which are equal or greater than those achieved by VLPs plus adjuvant in a liquid formulation. We conclude that in situ gelation of the GelVac dry powder formulation at nasal mucosal surfaces delays mucociliary clearance and thereby prolongs VLP antigen exposure to immune effector sites.

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.

An intranasally delivered Toll-like receptor 7 agonist elicits robust systemic and mucosal responses to Norwalk virus-like particles.

Norwalk virus (NV) is an enteric pathogen from the genus Norovirus and a major cause of nonbacterial gastroenteritis in humans. NV virus-like particles (VLPs) are known to elicit systemic and mucosal immune responses when delivered nasally; however, the correlates of immune protection are unknown, and codelivery with a safe and immunogenic mucosal adjuvant may enhance protective anti-NV immune responses. Resiquimod (R848), an imidazoquinoline-based Toll-like receptor 7 and/or 8 (TLR7/8) agonist, is being evaluated as an adjuvant in FDA-approved clinical vaccine trials. As such, we evaluated the adjuvant activity of two imidazoquinoline-based TLR7 and TLR7/8 agonists when codelivered intranasally with plant-derived NV VLPs. We also compared the activity of these agonists to the gold standard mucosal adjuvant, cholera toxin (CT). Our results indicate that codelivery with the TLR7 agonist, gardiquimod (GARD), induces NV VLP-specific serum IgG and IgG isotype responses and mucosal IgA responses in the gastrointestinal, respiratory, and reproductive tracts that are superior to those induced by R848 and comparable to those induced by the mucosal adjuvant CT. This study supports the continued investigation of GARD as a mucosal adjuvant for NV VLPs and possible use for other VLP-based vaccines for which immune responses at distal mucosal sites (e.g., respiratory and reproductive tracts) are desired.

HIV-1 neutralization profile and plant-based recombinant expression of actinohivin, an Env glycan-specific lectin devoid of T-cell mitogenic activity.

The development of a topical microbicide blocking the sexual transmission of HIV-1 is urgently needed to control the global HIV/AIDS pandemic. The actinomycete-derived lectin actinohivin (AH) is highly specific to a cluster of high-mannose-type glycans uniquely found on the viral envelope (Env). Here, we evaluated AH's candidacy toward a microbicide in terms of in vitro anti-HIV-1 activity, potential side effects, and recombinant producibility. Two validated assay systems based on human peripheral blood mononuclear cell (hPBMC) infection with primary isolates and TZM-bl cell infection with Env-pseudotyped viruses were employed to characterize AH's anti-HIV-1 activity. In hPMBCs, AH exhibited nanomolar neutralizing activity against primary viruses with diverse cellular tropisms, but did not cause mitogenicity or cytotoxicity that are often associated with other anti-HIV lectins. In the TZM-bl-based assay, AH showed broad anti-HIV-1 activity against clinically-relevant, mucosally transmitting strains of clades B and C. By contrast, clade A viruses showed strong resistance to AH. Correlation analysis suggested that HIV-1's AH susceptibility is significantly linked to the N-glycans at the Env C2 and V4 regions. For recombinant (r)AH expression, we evaluated a tobacco mosaic virus-based system in Nicotiana benthamiana plants as a means to facilitate molecular engineering and cost-effective mass production. Biochemical analysis and an Env-mediated syncytium formation assay demonstrated high-level expression of functional rAH within six days. Taken together, our study revealed AH's cross-clade anti-HIV-1 activity, apparent lack of side effects common to lectins, and robust producibility using plant biotechnology. These findings justify further efforts to develop rAH toward a candidate HIV-1 microbicide.

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.

Development and characterization of a three-dimensional organotypic human vaginal epithelial cell model.

We have developed an in vitro human vaginal epithelial cell (EC) model using the innovative rotating wall vessel (RWV) bioreactor technology that recapitulates in vivo structural and functional properties, including a stratified squamous epithelium with microvilli, tight junctions, microfolds, and mucus. This three-dimensional (3-D) vaginal model provides a platform for high-throughput toxicity testing of candidate microbicides targeted to combat sexually transmitted infections, effectively complementing and extending existing testing systems such as surgical explants or animal models. Vaginal ECs were grown on porous, collagen-coated microcarrier beads in a rotating, low fluid-shear environment; use of RWV bioreactor technology generated 3-D vaginal EC aggregates. Immunofluorescence and scanning and transmission electron microscopy confirmed differentiation and polarization of the 3-D EC aggregates among multiple cell layers and identified ultrastructural features important for nutrient absorption, cell-cell interactions, and pathogen defense. After treatment with a variety of toll-like receptor (TLR) agonists, cytokine production was quantified by cytometric bead array, confirming that TLRs 2, 3, 5, and 6 were expressed and functional. The 3-D vaginal aggregates were more resistant to nonoxynol-9 (N-9), a contraceptive and previous microbicide candidate, when compared to two-dimensional monolayers of the same cell line. A dose-dependent production of tumor necrosis factor-related apoptosis-inducing ligand and interleukin-1 receptor antagonist, biomarkers of cervicovaginal inflammation, correlated to microbicide toxicity in the 3-D model following N-9 treatment. These results indicate that this 3-D vaginal model could be used as a complementary tool for screening microbicide compounds for safety and efficacy, thus improving success in clinical trials.

A DNA replicon system for rapid high-level production of virus-like particles in plants.

Recombinant virus-like particles (VLPs) represent a safe and effective vaccine strategy. We previously described a stable transgenic plant system for inexpensive production and oral delivery of VLP vaccines. However, the relatively low-level antigen accumulation and long-time frame to produce transgenic plants are the two major roadblocks in the practical development of plant-based VLP production. In this article, we describe the optimization of geminivirus-derived DNA replicon vectors for rapid, high-yield plant-based production of VLPs. Co-delivery of bean yellow dwarf virus (BeYDV)-derived vector and Rep/RepA-supplying vector by agroinfiltration of Nicotiana benthamiana leaves resulted in efficient replicon amplification and robust protein production within 5 days. Co-expression of the P19 protein of tomato bush stunt virus, a gene silencing inhibitor, further enhanced VLP accumulation by stabilizing the mRNA. With this system, hepatitis B core antigen (HBc) and Norwalk virus capsid protein (NVCP) were produced at 0.80 and 0.34 mg/g leaf fresh weight, respectively. Sedimentation analysis and electron microscopy of transiently expressed antigens verified the efficient assembly of VLPs. Furthermore, a single replicon vector containing a built-in Rep/RepA cassette without P19 drove protein expression at similar levels as the three-component system. These results demonstrate the advantages of fast and high-level production of VLP-based vaccines using the BeYDV-derived DNA replicon system for transient expression in plants.

An efficient plant viral expression system generating orally immunogenic Norwalk virus-like particles.

Virus-like particles (VLPs) derived from enteric pathogens like Norwalk virus (NV) are well suited to study oral immunization. We previously described stable transgenic plants that accumulate recombinant NV-like particles (rNVs) that were orally immunogenic in mice and humans. The transgenic approach suffers from long generation time and modest level of antigen accumulation. We now overcome these constraints with an efficient tobacco mosaic virus (TMV)-derived transient expression system using leaves of Nicotiana benthamiana. We produced properly assembled rNV at 0.8 mg/g leaf 12 days post-infection (dpi). Oral immunization of CD1 mice with 100 or 250 microg/dose of partially purified rNV elicited systemic and mucosal immune responses. We conclude that the plant viral transient expression system provides a robust research tool to generate abundant quantities of rNV as enriched, concentrated VLP preparations that are orally immunogenic.

Synthesis and assembly of Escherichia coli heat-labile enterotoxin B subunit in transgenic lettuce (Lactuca sativa).

Escherichia coli heat-labile enterotoxin B subunit (LTB) strongly induces immune responses and can be used as an adjuvant for co-administered antigens. Synthetic LTB (sLTB) based on optimal codon usage by plants was introduced into lettuce cells (Lactuca sativa) by Agrobacterium tumefaciens-mediated transformation methods. The sLTB gene was detected in the genomic DNA of transgenic lettuce leaf cells by PCR DNA amplification. Synthesis and assembly of the sLTB protein into oligomeric structures of pentameric size was observed in transgenic plant extracts using Western blot analysis. The binding of sLTB pentamers to intestinal epithelial cell membrane glycolipid receptors was confirmed by G(M1)-ganglioside enzyme-linked immunosorbent assay (G(M1)-ELISA). Based on the results of ELISA, sLTB protein comprised approximately 1.0-2.0% of total soluble protein in transgenic lettuce leaf tissues. The synthesis and assembly of sLTB monomers into biologically active oligomers in transgenic lettuce leaf tissues demonstrates the feasibility of the use of edible plant-based vaccines consumed in the form of raw plant materials to induce mucosal immunity.

Induction of protective immune responses against the challenge of Actinobacillus pleuropneumoniae by the oral administration of transgenic tobacco plant expressing ApxIIA toxin from the bacteria.

Actinobacillus pleuropneumoniae is the causative agent of porcine pleuropneumonia. Among the virulence factors, ApxIIA, a bacterial exotoxin, is reportedly expressed in many serotypes and is considered as a candidate for the development of a vaccine against the bacterial infection. Previously, we isolated a field strain of A. pleuropneumoniae serotype 2 in Korea and characterized its exotoxins to develop an oral vaccine. In this study, we initially confirmed the immunogenicity of ApxIIA expressed in Escherichia coli. We then developed transgenic tobacco expressing ApxIIA and tested its efficacy to induce a protective immune response against A. pleuropneumoniae infection after oral administration of the plant powder. We observed that protective immune responses were induced in mice after oral administration of the plant powder once a week for 4 weeks. Immunoassays revealed that the levels of antigen-specific immunoglobulin G against ApxIIA increased in mice that were fed a powder made from the transgenic plant, but not in mice fed a powder made from wild-type tobacco. Additionally, mice fed the transgenic plant powder were protected from an injection of a lethal dose of A. pleuropneumoniae. These results support that the transgenic plant may be a suitable candidate for an oral vaccine that could be used effectively against A. pleuropneumoniae infection.

WHO informal consultation on scientific basis for regulatory evaluation of candidate human vaccines from plants, Geneva, Switzerland, 24-25 January 2005.

In January 2005, WHO convened a meeting of leading experts in plant-derived vaccines and experts from regulatory authorities for an informal discussion on the state-of-the-art and to analyse whether specific guidance might be needed for plant-derived vaccines that is not yet provided by regulatory authorities. After a series of individual presentations, a general discussion was held to obtain a consensus on the need for further guidance. Both the presentations and the conclusions are presented. The meeting concluded that existing guidelines for the development, evaluation, and use of vaccines made by traditional methods can be applied to plant-derived vaccines. For plant-derived vaccines some specific issues will have to be addressed. These include, but are not restricted to, containment of the plants including disposal of waste materials. It was noted that plant-derived vaccines have been produced and clinically tested under US investigational new drug application, and all applicable regulatory and good manufacturing practice requirements are in place for this type of product. An innovator wishing to bring a plant-derived vaccine to market should consult closely with regulatory authorities to ensure that all appropriate studies are undertaken.

Production of rotavirus-like particles in tomato (Lycopersicon esculentum L.) fruit by expression of capsid proteins VP2 and VP6 and immunological studies.

A number of different antigens have been successfully expressed in transgenic plants, and some are currently being evaluated as orally delivered vaccines. Here we report the successful expression of rotavirus capsid proteins VP2 and VP6 in fruits of transgenic tomato plants. By western blot analysis, using specific antibodies, we determined that the VP2 and VP6 produced in plants have molecular weights similar to those found in native rotavirus. The plant-synthesized VP6 protein retained the capacity to form trimers. We were able to recover rotavirus virus-like particles from tomato fruit (i.e., tomatoes) by centrifugation on a sucrose cushion and to visualize them by electron microscopy. This result indicated that VP2/VP6 can self-assemble into virus-like particles (VLPs) in plant cells, even though only a small proportion of VP2/VP6 assembled into VLPs. To investigate immunogenicity, adult mice were immunized intraperitoneally (i.p.) three times with a protein extract from a transgenic tomatoes in adjuvant. We found that the transgenic tomato extract induced detectable levels of anti-rotavirus antibodies in serum; however, we did not determine the contribution of either the free rotavirus proteins or the VLPs to the induction of the antibody response. These results suggest the potential of plant-based rotavirus VLPs for the development of a vaccine against rotavirus infection.

Protection conferred by recombinant Yersinia pestis antigens produced by a rapid and highly scalable plant expression system.

Plague is still an endemic disease in different regions of the world. Increasing reports of incidence, the discovery of antibiotic resistance strains, and concern about a potential use of the causative bacteria Yersinia pestis as an agent of biological warfare have highlighted the need for a safe, efficacious, and rapidly producible vaccine. The use of F1 and V antigens and the derived protein fusion F1-V has shown great potential as a protective vaccine in animal studies. Plants have been extensively studied for the production of pharmaceutical proteins as an inexpensive and scalable alternative to common expression systems. In the current study the recombinant plague antigens F1, V, and fusion protein F1-V were produced by transient expression in Nicotiana benthamiana by using a deconstructed tobacco mosaic virus-based system that allowed very rapid and extremely high levels of expression. All of the plant-derived purified antigens, administered s.c. to guinea pigs, generated systemic immune responses and provided protection against an aerosol challenge of virulent Y. pestis.

Rapid, high-level production of hepatitis B core antigen in plant leaf and its immunogenicity in mice.

Hepatitis B core antigen (HBc or HBcAg) self-assembles into capsid particles and is extremely immunogenic. HBc has been extensively studied for its production in various expression systems and for the use of HBc particles for high-density, immunogenic presentation of foreign epitopes. Here we reported the high-level transient expression of HBc in plant leaf and its immunogenicity in mice. By using a novel plant viral expression system, HBc was produced in Nicotiana benthamiana leaves at levels up to 7.14% of total soluble protein (TSP) or 2.38 milligrams HBc per gram of fresh weight at 7 days post-infection (dpi). Plant-derived HBc (p-HBc) assembled into virus-like particles (VLPs) as revealed by sucrose gradients and electron microscopy. Partially purified p-HBc stimulated strong serum antibody responses in mice as Escherichia coli-derived HBc upon intraperitoneal (i.p.) injection. Furthermore, mice immunized mucosally (orally and intranasally) with p-HBc in the absence of adjuvants also developed HBc-specific serum IgG as well as intestinal IgA. Taken together, our results indicate the potential usefulness of p-HBc-VLP as a carrier for immunogenic presentation and mucosal delivery of foreign epitopes.

Virus-like particle expression and assembly in plants: hepatitis B and Norwalk viruses.

Expression of vaccine antigens in plants and delivery via ingestion of transgenic plant material has shown promise in numerous pre-clinical animal studies and in a few clinical trials. A number of different viral antigens have been tested, and among the most promising are those that can assemble virus-like particles (VLP), which mimic the form of authentic virions and display neutralizing antibody epitopes. We have extensively studied plant expression, VLP assembly, and immunogenicity of hepatitis B surface antigen (HBsAg) and Norwalk virus capsid protein (NVCP). The HBsAg small protein (S protein) was found by TEM to assemble tubular membrane complexes derived from endoplasmic reticulum in suspension cultured cells of tobacco and soybean, and in potato leaf and tuber tissues. The potato material was immunogenic in mice upon delivery by ingestion. Here we describe the plant expression and immunogenicity of HBsAg middle protein (M protein or pre-S2 + S) which contains additional 55 amino acid pre-S2 region at N-terminus of the S protein. Plant-derived recombinant M protein provoked stronger serum antibody responses against HBsAg than did S protein when injected systemically in mice. We discuss implications for use of fusion proteins for enhanced immunogenicity and mucosal targeting of HBsAg, as well as delivery of heterologous fused antigens. NVCP expressed in plants assembled 38 nm virion-size icosahedral (T = 3) VLP, similar to those produced in insect cells. The VLP stimulated serum IgG and IgA responses in mice and humans when they were delivered by ingestion of fresh potato tuber. Here we show that freeze-drying of transgenic NVCP tomato fruit yielded stable preparations that stimulated excellent IgG and IgA responses against NVCP when fed to mice. However, the predominant VLP form in tomato fruit was the small 23 nm particle also observed in insect cell-derived NVCP.