R. Mark Jones

A Plant-Produced Pfs25 VLP Malaria Vaccine Candidate Induces Persistent Transmission Blocking Antibodies against Plasmodium falciparum in Immunized Mice

Malaria transmission blocking vaccines (TBVs) are considered an effective means to control and eventually eliminate malaria. The Pfs25 protein, expressed predominantly on the surface of the sexual and sporogonic stages of Plasmodium falciparum including gametes, zygotes and ookinetes, is one of the primary targets for TBV. It has been demonstrated that plants are an effective, highly scalable system for the production of recombinant proteins, including virus-like particles (VLPs). We engineered VLPs (Pfs25-CP VLP) comprising Pfs25 fused to the Alfalfa mosaic virus coat protein (CP) and produced these non-enveloped hybrid VLPs in Nicotiana benthamiana plants using a Tobacco mosaic virus-based ‘launch’ vector. Purified Pfs25-CP VLPs were highly consistent in size (19.3±2.4 nm in diameter) with an estimated 20–30% incorporation of Pfs25 onto the VLP surface. Immunization of mice with one or two doses of Pfs25-CP VLPs plus Alhydrogel® induced serum antibodies with complete transmission blocking activity through the 6 month study period. These results support the evaluation of Pfs25-CP VLP as a potential TBV candidate and the feasibility of the ‘launch’ vector technology for the production of VLP-based recombinant vaccines against infectious diseases.

Neutralization of Botulinum Neurotoxin by a Human Monoclonal Antibody Specific for the Catalytic Light Chain

Background Botulinum neurotoxins (BoNT) are a family of category A select bioterror agents and the most potent biological toxins known. Cloned antibody therapeutics hold considerable promise as BoNT therapeutics, but the therapeutic utility of antibodies that bind the BoNT light chain domain (LC), a metalloprotease that functions in the cytosol of cholinergic neurons, has not been thoroughly explored. Methods and Findings We used an optimized hybridoma method to clone a fully human antibody specific for the LC of serotype A BoNT (BoNT/A). The 4LCA antibody demonstrated potent in vivo neutralization when administered alone and collaborated with an antibody specific for the HC. In Neuro-2a neuroblastoma cells, the 4LCA antibody prevented the cleavage of the BoNT/A proteolytic target, SNAP-25. Unlike an antibody specific for the HC, the 4LCA antibody did not block entry of BoNT/A into cultured cells. Instead, it was taken up into synaptic vesicles along with BoNT/A. The 4LCA antibody also directly inhibited BoNT/A catalytic activity in vitro. Conclusions An antibody specific for the BoNT/A LC can potently inhibit BoNT/A in vivo and in vitro, using mechanisms not previously associated with BoNT-neutralizing antibodies. Antibodies specific for BoNT LC may be valuable components of an antibody antidote for BoNT exposure.

A novel plant-produced Pfs25 fusion subunit vaccine induces long-lasting transmission blocking antibody responses.

Malaria transmission blocking vaccines (TBV) directed against proteins expressed on sexual stages of Plasmodium falciparum in the mosquito midgut are considered an effective means to reduce malaria transmission. Antibodies induced by TBV block sporogonic development in the mosquito, and thus transmission to the next human host. The Pfs25 protein, expressed on the surface of gametes, zygotes and ookinetes, is one of the primary targets for TBV development. Using a plant virus-based transient expression system, we have successfully produced Pfs25 fused to a modified lichenase (LicKM) carrier in Nicotiana benthamiana, purified and characterized the protein (Pfs25-FhCMB), and evaluated this vaccine candidate in animal models for the induction of transmission blocking antibodies. Soluble Pfs25-FhCMB was expressed in plants at a high level, and induced transmission blocking antibodies that persisted for up to 6 months post immunization in mice and rabbits. These data demonstrate the potential of the new malaria vaccine candidate and also support feasibility of expressing Plasmodium antigens in a plant-based system.

Production of non‐glycosylated recombinant proteins in Nicotiana benthamiana plants by co‐expressing bacterial PNGase F

Application of tools of molecular biology and genomics is increasingly leading towards the development of recombinant protein-based biologics. As such, it is leading to an increased diversity of targets that have important health applications and require more flexible approaches for expression because of complex post-translational modifications. For example, Plasmodium parasites may have complex post-translationally modified proteins such as Pfs48/45 that do not carry N-linked glycans (Exp. Parasitol. 1998; 90, 165.) but contain potential N-linked glycosylation sites that can be aberrantly glycosylated during expression in mammalian and plant systems. Therefore, it is important to develop strategies for producing non-glycosylated forms of these targets to preserve biological activity and native conformation. In this study, we are describing in vivo deglycosylation of recombinant N-glycosylated proteins as a result of their transient co-expression with bacterial PNGase F (Peptide: N-glycosidase F). In addition, we show that the recognition of an in vivo deglycosylated plant-produced malaria vaccine candidate, Pfs48F1, by monoclonal antibodies I, III and V raised against various epitopes (I, III and V) of native Pfs48/45 of Plasmodium falciparum, was significantly stronger compared to that of the glycosylated form of plant-produced Pfs48F1. To our knowledge, neither in vivo enzymatic protein deglycosylation has been previously achieved in any eukaryotic system, including plants, nor has bacterial PNGase F been expressed in the plant system. Thus, here, we report for the first time the expression in plants of an active bacterial enzyme PNGase F and the production of recombinant proteins of interest in a non-glycosylated form.

Hybridoma populations enriched for affinity-matured human IgGs yield high-affinity antibodies specific for botulinum neurotoxins.

The affinity-matured human antibody repertoire may be ideal as a source for antibody therapeutics against infectious diseases and bioterror agents. Hybridoma methods for cloning these antibodies have many potential advantages, including convenience, high-yield antibody expression, and the ability to capture the antibodies in their native configurations. However, they have been hindered by hybridoma instability and limited accessibility of antigen-specific, class-switched human B-cells. Here, we describe an efficient, three-step method that uses human peripheral blood B-cells to produce stable hybridoma populations that are highly-enriched for affinity-matured human IgG antibodies. Peripheral blood mononuclear cells (PBMCs) are (a) selected for expression of CD27, a marker of post-germinal center B-cells, (b) cultured in vitro to promote B-cell proliferation and class-switching, and (c) fused to a genetically modified myeloma cell line. Using this strategy, we cloned 5 IgG antibodies that bind botulinum neurotoxins (BoNT), the causes of the food-borne paralytic illness, botulism, and Category A Select Bioterror agents. Two of these antibodies bind BoNT with low picomolar affinities. One (30B) is the first high-affinity human antibody to bind serotype B BoNT, and another (6A) is able to neutralize a lethal dose of serotype A BoNT in vivo in pre- and post-exposure models. This optimized hybridoma method will broadly enable access to the native human antibody repertoire.

A plant-produced H1N1 trimeric hemagglutinin protects mice from a lethal influenza virus challenge.

The increased worldwide awareness of seasonal and pandemic influenza, including pandemic H1N1 virus, has stimulated interest in the development of economic platforms for rapid, large-scale production of safe and effective subunit vaccines. In recent years, plants have demonstrated their utility as such a platform and have been used to produce vaccine antigens against various infectious diseases. Previously, we have produced in our transient plant expression system a recombinant monomeric hemagglutinin (HA) protein (HAC1) derived from A/California/04/09 (H1N1) strain of influenza virus and demonstrated its immunogenicity and safety in animal models and human volunteers. In the current study, to mimic the authentic HA structure presented on the virus surface and to improve stability and immunogenicity of the HA antigen, we generated trimeric HA by introducing a trimerization motif from a heterologous protein into the HA sequence. Here, we describe the engineering, production in Nicotiana benthamiana plants, and characterization of the highly purified recombinant trimeric HA protein (tHA-BC) from A/California/04/09 (H1N1) strain of influenza virus. The results demonstrate the induction of serum hemagglutination inhibition antibodies by tHA-BC and its protective efficacy in mice against a lethal viral challenge. In addition, the immunogenic and protective doses of tHA-BC were much lower compared with monomeric HAC1. Further investigation into the optimum vaccine dose and/or regimen as well as the stability of trimerized HA is necessary to determine whether trimeric HA is a more potent vaccine antigen than monomeric HA.

A Human Monoclonal Antibody that Binds Serotype A Botulinum Neurotoxin

Monoclonal antibodies have demonstrated significant potential as therapeutics for botulinum neurotoxin exposures. We previously described a hybridoma method for cloning native human antibodies that uses a murine myeloma cell line that ectopically expresses the human telomerase catalytic subunit gene (hTERT) and the murine interleukin-6 gene (mIL-6). Here we describe a heterohybridoma cell line that ectopically expresses mIL-6 and hTERT and has improved stability of hTERT expression. We fused this cell line to human peripheral blood B cells from a subject who had received the botulinum toxoid vaccine, cloning a high-affinity antibody (13A) specific for serotype A botulinum neurotoxin (BoNT/A). The 13A antibody is an affinity-matured, post-germinal center IgG(1) lambda antibody that has partial neutralization activity in vivo. 13A binds an epitope on BoNT/A that overlaps the binding epitope of an IgG antibody previously shown to fully neutralize a lethal dose of BoNT/A in vivo. The 13A antibody may be useful for diagnostic testing or for incorporation into an oligoclonal therapeutic to counteract BoNT/A exposure.

Immunogenicity of H1N1 influenza virus-like particles produced in Nicotiana benthamiana

The H1N1 influenza pandemic of 2009 stimulated interest in developing safe and effective subunit influenza vaccines using rapid and cost-effective recombinant technologies that can avoid dependence on hens’ eggs supply and live viruses for production. Among alternative approaches to subunit vaccine development, virus-like particles (VLPs) represent an attractive strategy due to their safety and immunogenicity. Previously, we have produced a recombinant monomeric hemagglutinin (HA) protein derived from the A/California/04/09 (H1N1) strain of influenza virus in a plant-based transient expression system and demonstrated immunogenicity and safety of this monomeric HA in animal models and human volunteers. In an effort to produce higher potency influenza vaccine in plants, we have designed and generated enveloped VLPs using the ectodomain of HA from the A/California/04/09 strain and heterologous sequences. The resulting H1 HA VLPs (HAC-VLPs) elicited robust hemagglutination inhibition antibody responses in mice at doses lower than 1 µg in the presence or absence of Alhydrogel adjuvant. These results suggest enhanced immunogenicity of recombinant HA in the form of an enveloped VLP over soluble antigen.

Plant-produced transmission blocking Plasmodium falciparum Pfs25 subunit and VLP based vaccine candidates

Malaria is a serious mosquito-borne disease caused by a protozoan parasite. Vaccines can target different stages of the pathogen’s life cycle. Transmission blocking vaccines target mosquito stages of the parasite life cycle, and will support eradication programs to ease the disease burden at the population level. Pfs25 is a sexual stage protein of Plasmodium falciparum which is found on the surface of the parasite zygote as it develops in the mosquito midgut. Antibodies against this protein block zygote development, and as a result block transmission to the next human host. Pfs25 was successfully expressed in our plant-based launch vector system as a fusion to the lichenase carrier molecule and as a fusion to the Alfalfa mosaic virus coat protein (AIMV CP), and in each case was purified to a high level of homogeneity. The resulting Pfs25-lichenase and Pfs25-AIMVCP antigens have undergone extensive biochemical characterization and dose-ranging studies in pre-clinical animal models, where both antigens induced transmission blocking antibodies. These data demonstrate the feasibility of expressing Plasmodium antigens in a plant-based system for the economical production of a transmission-blocking vaccine against malaria.

Formulation development of a plant-derived h1n1 influenza vaccine containing purified recombinant hemagglutinin antigen

Influenza is a prevalent, highly contagious and sometimes fatal respiratory disease. Vaccination provides an effective approach to control the disease, but because of frequent changes in the structure of the major surface proteins, there is great need for a technology that permits rapid preparation of new forms of the vaccine each year in sufficient quantities. Recently, using a safe, simple, time- and cost-effective plant viral vector-based transient expression system, the hemagglutinin antigen of H1N1 influenza A strain (HAC1), an H1N1 influenza vaccine candidate, has been produced in Nicotiana benthamiana plants. As a step toward the generation of a commercially viable subunit influenza vaccine, we developed HAC1 formulations in the presence and absence of an aluminum salt adjuvant (Alhydrogel®), analyzed their properties, and assessed immunogenicity in an animal model. Biophysical properties of HAC1 were evaluated using several spectroscopic and light scattering techniques as a function of pH and temperature combined with data analysis using an empirical phase diagram approach. Excipients that were potent stabilizers of the recombinant protein were identified using intrinsic fluorescence spectroscopy. The adsorptive capacity and thermal stability of the protein on the surface of Alhydrogel® were then examined in the presence and absence of selected stabilizers using UV absorbance after centrifugation and intrinsic fluorescence spectroscopy, respectively. Immunogenicity studies conducted in mice demonstrated that the highest level of serum immune responses (hemagglutination-inhibiting antibody titers), with a 100% seropositive rates, were induced by HAC1 in the presence of Alhydrogel®, and this response was elicited regardless of the solution conditions of the formulation.