Basic leucine zipper transcription activators - tools to improve production and quality of human erythropoietin in Nicotiana benthamiana.

Human erythropoietin (hEPO) is one of the most in-demand biopharmaceuticals, however, its production is challenging. When produced in a plant expression system, hEPO results in extensive plant tissue damage and low expression. It is demonstrated that the modulation of the plant protein synthesis machinery enhances hEPO production. Co-expression of basic leucine zipper transcription factors with hEPO prevents plant tissue damage, boosts expression, and increases hEPO solubility. bZIP28 co-expression up-regulates genes associated with the unfolded protein response, indicating that the plant tissue damage caused by hEPO expression is due to the native protein folding machinery being overwhelmed and that this can be overcome by co-expressing bZIP28.

Engineering of a plant-produced virus-like particle to improve the display of the Plasmodium falciparum Pfs25 antigen and transmission-blocking activity of the vaccine candidate.

Malaria kills around 409,000 people a year, mostly children under the age of five. Malaria transmission-blocking vaccines work to reduce malaria prevalence in a community and have the potential to be part of a multifaceted approach required to eliminate the parasites causing the disease. Pfs25 is a leading malaria transmission-blocking antigen and has been successfully produced in a plant expression system as both a subunit vaccine and as a virus-like particle. This study demonstrates an improved version of the virus-like particle antigen display molecule by eliminating known protease sites from the prior A85 variant. This re-engineered molecule, termed B29, displays three times the number of Pfs25 antigens per virus-like particle compared to the original Pfs25 virus-like particle. An improved purification scheme was also developed, resulting in a substantially higher yield and improved purity. The molecule was evaluated in a mouse model and found to induce improved transmission-blocking activity at lower doses and longer durations than the original molecule.

Safety and immunogenicity of a plant-produced Pfs25 virus-like particle as a transmission blocking vaccine against malaria: A Phase 1 dose-escalation study in healthy adults.

Malaria continues to be one of the world's most devastating infectious tropical diseases, and alternative strategies to prevent infection and disease spread are urgently needed. These strategies include the development of effective vaccines, such as malaria transmission blocking vaccines (TBV) directed against proteins found on the sexual stages of Plasmodium falciparum parasites present in the mosquito midgut. The Pfs25 protein, which is expressed on the surface of gametes, zygotes and ookinetes, has been a primary target for TBV development. One such vaccine strategy based on Pfs25 is a plant-produced malaria vaccine candidate engineered as a chimeric non-enveloped virus-like particle (VLP) comprising Pfs25 fused to the Alfalfa mosaic virus coat protein. This Pfs25 VLP-FhCMB vaccine candidate has been engineered and manufactured in Nicotiana benthamiana plants at pilot plant scale under current Good Manufacturing Practice guidelines. The safety, reactogenicity and immunogenicity of Pfs25 VLP-FhCMB was assessed in healthy adult volunteers. This Phase 1, dose escalation, first-in-human study was designed primarily to evaluate the safety of the purified plant-derived Pfs25 VLP combined with Alhydrogel® adjuvant. At the doses tested in this Phase 1 study, the vaccine was generally shown to be safe in healthy volunteers, with no incidence of vaccine-related serious adverse events and no evidence of any dose-limiting or dose-related toxicity, demonstrating that the plant-derived Pfs25 VLP-FhCMB vaccine had an acceptable safety and tolerability profile. In addition, although the vaccine did induce Pfs25-specific IgG in vaccinated patients in a dose dependent manner, the transmission reducing activity of the antibodies generated were weak, suggesting the need for an alternative vaccine adjuvant formulation. This study was registered at www.ClinicalTrials.gov under reference identifier NCT02013687.

Plant-Produced Subunit Vaccine Candidates against Yellow Fever Induce Virus Neutralizing Antibodies and Confer Protection against Viral Challenge in Animal Models.

Yellow fever (YF) is a viral disease transmitted by mosquitoes and endemic mostly in South America and Africa with 20-50% fatality. All current licensed YF vaccines, including YF-Vax® (Sanofi-Pasteur, Lyon, France) and 17DD-YFV (Bio-Manguinhos, Rio de Janeiro, Brazil), are based on live attenuated virus produced in hens' eggs and have been widely used. The YF vaccines are considered safe and highly effective. However, a recent increase in demand for YF vaccines and reports of rare cases of YF vaccine-associated fatal adverse events have provoked interest in developing a safer YF vaccine that can be easily scaled up to meet this increased global demand. To this point, we have engineered the YF virus envelope protein (YFE) and transiently expressed it in Nicotiana benthamiana as a stand-alone protein (YFE) or as fusion to the bacterial enzyme lichenase (YFE-LicKM). Immunogenicity and challenge studies in mice demonstrated that both YFE and YFE-LicKM elicited virus neutralizing (VN) antibodies and protected over 70% of mice from lethal challenge infection. Furthermore, these two YFE-based vaccine candidates induced VN antibody responses with high serum avidity in nonhuman primates and these VN antibody responses were further enhanced after challenge infection with the 17DD strain of YF virus. These results demonstrate partial protective efficacy in mice of YFE-based subunit vaccines expressed in N. benthamiana. However, their efficacy is inferior to that of the live attenuated 17DD vaccine, indicating that formulation development, such as incorporating a more suitable adjuvant, may be required for product development.

Purification and immunogenicity of hemagglutinin from highly pathogenic avian influenza virus H5N1 expressed in Nicotiana benthamiana.

Highly pathogenic avian influenza (HPAI) H5N1 is an ongoing global health concern due to its severe sporadic outbreaks in Asia, Africa and Europe, which poses a potential pandemic threat. The development of safe and cost-effective vaccine candidates for HPAI is considered the best strategy for managing the disease and addressing the pandemic preparedness. The most potential vaccine candidate is the antigenic determinant of influenza A virus, hemagglutinin (HA). The present research was aimed at developing optimized expression in Nicotiana benthamiana and protein purification process for HA from the Malaysian isolate of H5N1 as a vaccine antigen for HPAI H5N1. Expression of HA from the Malaysian isolate of HPAI in N. benthamiana was confirmed, and more soluble protein was expressed as truncated HA, the HA1 domain over the entire ectodomain of HA. Two different purification processes were evaluated for efficiency in terms of purity and yield. Due to the reduced yield, protein degradation and length of the 3-column purification process, the 2-column method was chosen for target purification. Purified HA1 was found immunogenic in mice inducing H5 HA-specific IgG and a hemagglutination inhibition antibody. This paper offers an alternative production system of a vaccine candidate against a locally circulating HPAI, which has a regional significance.

Engineering and expression of a RhoA peptide against respiratory syncytial virus infection in plants.

MAIN CONCLUSION : A RhoA-derived peptide fused to carrier molecules from plants showed enhanced biological activity of in vitro assays against respiratory syncytial virus compared to the RhoA peptide alone or the synthetic RhoA peptide. A RhoA-derived peptide has been reported for over a decade as a potential inhibitor of respiratory syncytial virus (RSV) infection both in vitro and in vivo and is anticipated to be a promising alternative to monoclonal antibody-based therapy against RSV infection. However, there are several challenges to furthering development of this antiviral peptide, including improvement in the peptide's bioavailability, development of an efficient delivery system and identification of a cost-effective production platform. In this study, we have engineered a RhoA peptide as a genetic fusion to two carrier molecules, either lichenase (LicKM) or the coat protein (CP) of Alfalfa mosaic virus. These constructs were introduced into Nicotiana benthamiana plants using a tobacco mosaic virus-based expression vector and targets purified. The results demonstrated that the RhoA peptide fusion proteins were efficiently expressed in N. benthamiana plants, and that two of the resulting fusion proteins, RhoA-LicKM and RhoA2-FL-d25CP, inhibited RSV growth in vitro by 50 and 80 %, respectively. These data indicate the feasibility of transient expression of this biologically active antiviral RhoA peptide in plants and the advantage of using a carrier molecule to enhance target expression and efficacy.

Novel phenotype in beagle dogs characterized by skin response to compound 48/80 focusing on skin mast cell degranulation.

Beagle dogs have long been employed in toxicology studies and as skin disease models. Compared with other experimental animal species, they are known to be susceptible to skin responses, such as rashes, from exposure to various chemical compounds. Here, a unique dog phenotype was identified that showed no skin response to compound 48/80, a mast cell degranulating agent. Although the skin responses to intradermal injection of polyoxyethylene castor oil derivative (HCO-60, a nonionic detergent), histamine dihydrochloride, concanavalin A (IgE receptor-mediated stimuli), or calcium ionophore A23187 were comparable in wild-type (WT) dogs and these nonresponder (NR) dogs, only the response to compound 48/80 was entirely absent from NR dogs. The skin mast cell density and histamine content per mast cell were histologically comparable between WT and NR dogs. By checking for skin responses to compound 48/80, NR dogs were found to exist at the proportion of 17-20% among four animal breeders. From retrospective analysis of in-house breeding histories, the NR phenotype appears to conform to the Mendelian pattern of recessive inheritance. The standard skin response in WT dogs developed at 2-4 months of age. In conclusion, this unique phenotype, typified by insensitivity in the compound 48/80-induced degranulation pathway in mast cells, has been widely retained by recessive inheritance in beagle dogs among general experimental animal breeders. The knowledge concerning this phenotype could lead to better utilization of dogs in studies and aid in model development.

Intranasal vaccination with a plant-derived H5 HA vaccine protects mice and ferrets against highly pathogenic avian influenza virus challenge.

Highly pathogenic avian influenza H5N1 infection remains a public health threat and vaccination is the best measure of limiting the impact of a potential pandemic. Mucosal vaccines have the advantage of eliciting immune responses at the site of viral entry, thereby preventing infection as well as further viral transmission. In this study, we assessed the protective efficacy of hemagglutinin (HA) from the A/Indonesia/05/05 (H5N1) strain of influenza virus that was produced by transient expression in plants. The plant-derived vaccine, in combination with the mucosal adjuvant (3',5')-cyclic dimeric guanylic acid (c-di-GMP) was used for intranasal immunization of mice and ferrets, before challenge with a lethal dose of the A/Indonesia/05/05 (H5N1) virus. Mice vaccinated with 15 µg or 5 µg of adjuvanted HA survived the viral challenge, while all control mice died within 10 d of challenge. Vaccinated animals elicited serum hemagglutination inhibition, IgG and IgA antibody titers. In the ferret challenge study, all animals vaccinated with the adjuvanted plant vaccine survived the lethal viral challenge, while 50% of the control animals died. In both the mouse and ferret models, the vaccinated animals were better protected from weight loss and body temperature changes associated with H5N1 infection compared with the non-vaccinated controls. Furthermore, the systemic spread of the virus was lower in the vaccinated animals compared with the controls. Results presented here suggest that the plant-produced HA-based influenza vaccine adjuvanted with c-di-GMP is a promising vaccine/adjuvant combination for the development of new mucosal influenza vaccines.

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.

Stabilization of HAC1 influenza vaccine by spray drying: formulation development and process scale-up.

PURPOSE: Stable vaccines with long shelf lives and reduced dependency on the cold chain are ideal for stockpiling and rapid deployment during public emergencies, including pandemics. Spray drying is a low-cost process that has potential to produce vaccines stable at a wide range of temperatures. Our aim was to develop a stable formulation of a recombinant H1N1 influenza hemagglutinin vaccine candidate and take it to pilot-scale spray-drying production. METHODS: Eight formulations containing different excipients were produced and assayed for antigen stability, powder characteristics, and immunogenicity after storage at a range of temperatures, resulting in the identification of four promising candidates. A pilot-scale spray-drying process was then developed for further testing of one formulation. RESULTS: The pilot-scale process was used to reproducibly manufacture three batches of the selected formulation with yields >90%. All batches had stable physical properties and in vitro potency for 6 months at temperatures from -20°C to +50°C. Formulations stored for 3 months elicited immunogenic responses in mice equivalent to a frozen lot of bulk vaccine used as a stability control. CONCLUSIONS: This study demonstrates the feasibility of stabilizing subunit vaccines using a spray-drying process and the suitability of the process for manufacturing a candidate product.

A new adjuvanted nanoparticle-based H1N1 influenza vaccine induced antigen-specific local mucosal and systemic immune responses after administration into the lung.

Annually influenza virus infections are responsible for hospitalization and mortality, especially in high risk groups. Constant antigenic changes in seasonal influenza viruses resulted from antigenic shifts and antigenic drifts, enable emerging of novel virus subtypes that may reduce current vaccine efficacy and impose the continuous revision of vaccine component. Currently available vaccines are usually limited by their production processes in terms of rapid adaptation to new circulating subtypes in high quantities meeting the global demand. Thus, new approaches to rapidly manufacture high yields of influenza vaccines are required. New technologies to reach maximal protection with minimal vaccine doses also need to be developed. In this study, we evaluated the systemic and local immunogenicity of a new double-adjuvanted influenza vaccine administered at the site of infection, the respiratory tract. This vaccine combines a plant-produced H1N1 influenza hemagglutinin antigen (HAC1), a silica nanoparticle-based (SiO2) drug delivery system and the mucosal adjuvant candidate bis-(3',5')-cyclic dimeric guanosine monophosphate (c-di-GMP). Mice were vaccinated by intratracheal route with HAC1/SiO2 or HAC1/c-di-GMP (single-adjuvanted vaccine) or HAC1/SiO2/c-di-GMP (double-adjuvanted vaccine) and evaluated for target-specific immune responses, such as hemagglutination inhibition and hemagglutinin-specific IgG titers, as well as local antibody (IgG and IgA) titers in the bronchoalveolar lavage (BAL). Furthermore, the HAC1-specific T-cell re-stimulation potential was assessed using precision-cut lung slices (PCLS) of vaccinated mice. The double-adjuvanted vaccine induced high systemic antibody responses comparable to the systemic vaccination control. In addition, it induced local IgG and IgA responses in the BAL. Furthermore, HAC1 induced a local T-cell response demonstrated by elevated IL-2 and IFN-γ levels in PCLS of c-di-GMP-vaccinated mice upon re-stimulation. Overall, the present study showed the potential of the double-adjuvanted vaccine to induce systemic humoral immune responses in intratracheally vaccinated mice. Furthermore, it induced a strong mucosal immune response, with evidence of antigen-primed T-cells in the lung.

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.

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.

A plant-based system for rapid production of influenza vaccine antigens.

BACKGROUND: Influenza virus is a globally important respiratory pathogen that causes a high degree of annual morbidity and mortality. Significant antigenic drift results in emergence of new, potentially pandemic, virus variants. The best prophylactic option for controlling emerging virus strains is to manufacture and administer pandemic vaccines in sufficient quantities and to do so in a timely manner without impacting the regular seasonal influenza vaccine capacity. Current, egg-based, influenza vaccine production is well established and provides an effective product, but has limited capacity and speed. OBJECTIVES: To satisfy the additional global demand for emerging influenza vaccines, high-performance cost-effective technologies need to be developed. Plants have a potential as an economic and efficient large-scale production platform for vaccine antigens. METHODS: In this study, a plant virus-based transient expression system was used to produce hemagglutinin (HA) proteins from the three vaccine strains used during the 2008-2009 influenza season, A/Brisbane/59/07 (H1N1), A/Brisbane/10/07 (H3N2), and B/Florida/4/06, as well as from the recently emerged novel H1N1 influenza A virus, A/California/04/09. RESULTS: The recombinant plant-based HA proteins were engineered and produced in Nicotiana benthamiana plants within 2 months of obtaining the genetic sequences specific to each virus strain. These antigens expressed at the rate of 400-1300 mg/kg of fresh leaf tissue, with >70% solubility. Immunization of mice with these HA antigens induced serum anti-HA IgG and hemagglutination inhibition antibody responses at the levels considered protective against these virus infections. CONCLUSIONS: These results demonstrate the feasibility of our transient plant expression system for the rapid production of influenza vaccine antigens.

An influenza N1 neuraminidase-specific monoclonal antibody with broad neuraminidase inhibition activity against H5N1 HPAI viruses.

H5N1 avian influenza continues to be a potential pandemic threat. Several vaccine candidates based on potentially pandemic influenza strains and antiviral drugs have been tested in preclinical and clinical studies. The data obtained so far have shown some promise, but have also revealed some shortcomings with both of these approaches. We have identified and characterized an H5N1 neuraminidasespecific monoclonal antibody which specifically inhibits N1 neuraminidase activity of highly pathogenic avian influenza (HPAI) strains from clades 1 and 2. We have also shown the protective efficacy of this antibody in animal challenge models using homologous virus. Specific and effective inhibition of N1 NA could make this mAb a useful therapeutic tool in the treatment of human infection, in particular with oseltamivirand zanamivir-resistant strains of HPAI.

Plant-based rapid production of recombinant subunit hemagglutinin vaccines targeting H1N1 and H5N1 influenza.

In 2009, a novel H1N1 swine influenza virus was isolated from infected humans in Mexico and the United States, and rapidly spread around the world. Another virus, a highly pathogenic avian influenza virus of the H5N1 subtype, identified by the World Health Organization as a potential pandemic threat in 1997, continues to be a significant risk. While vaccination is the preferred strategy for the prevention and control of influenza infections, the traditional egg-based approach to producing influenza vaccines does not provide sufficient capacity and adequate speed to satisfy global needs to combat newly emerging strains, seasonal or potentially pandemic. Significant efforts are underway to develop and implement new cell substrates with improved efficiency for influenza vaccine development and manufacturing. In recent years, plants have been used to produce recombinant proteins including subunit vaccines and antibodies. The main advantages of using plant systems for the production of vaccine antigens against influenza are their independence from pathogenic viruses, and cost and time efficiency. Here, we describe the large-scale production of recombinant hemagglutinin proteins from A/California/04/09 (H1N1) and A/Indonesia/05/05 (H5N1) strains of influenza virus in Nicotiana benthamiana plants, and their immunogenicity (serum hemagglutination inhibition and virus neutralizing antibodies), and safety in animal models. These results support the testing of these candidate vaccines in human volunteers and also the utility of our plant expression system for large-scale recombinant influenza vaccine production.

Specific transgene expression in HIV-infected cells using protease-cleavable transcription regulator.

Gene therapy is a promising strategy for the treatment of HIV infection, but cell specificity remains an issue. Recently we have developed a new concept for a drug or gene delivery system responding to cellular signals (D-RECS) to achieve cell-specific transgene expression using a non-viral polymer-based vehicle. According to this concept, intracellular signaling enzymes, which are activated specifically in target cells, are used to trigger transgene expression. We previously applied this concept to HIV-1 protease and showed that the recombinant protease could act as a suitable signal. Here we further developed this system to achieve highly specific transgene expression in HIV-infected cells. We prepared a polymeric gene regulator grafted with a cationic peptide containing the HIV-Tat peptide via a specific substrate for HIV-1 protease. The regulator formed a stable polyplex with the transgene, suppressing its transcription. HIV-1 protease cleaved the peptide and released the transgene, which was consequently expressed specifically in activated HIV-infected cells, but remained unreleased and inactive in uninfected cells. The validity of this approach was further confirmed by applying it to the CVB1 2A protease of coxsackievirus (Picornaviridae family). This strategy should be widely applicable for specific expression of a variety of therapeutic genes in virus-infected cells.

Inflammatory cell-specific transgene expression system responding to Ikappa-B kinase beta activation.

BACKGROUND: Control of inflammation is essential for the clinical management of many common human diseases. However, there are few generally applicable strategies to convert an abnormal intracellular signal into a gene expression that leads to normalization of the intracellular environment. Recently, we proposed a novel strategy termed D-RECS (i.e. drug or gene delivery system responding to cellular signals) to convert an intracellular signal to transgene expression. In the present study, we applied this concept to inflammatory cells using Ikappa-B kinase as a signal molecule that triggers the gene expression. METHODS: Candidate cationic substrates of Ikappa-B kinase (IKK)beta were synthesized and their reactivity was investigated. Then, polymers grafted with these peptides were prepared by radical polymerization. Polymer/DNA complexes (polyplexes) were prepared by mixing plasmid DNAs with the polymers. The behaviour of these polyplexes by adding IKKbeta was examined. Furthermore, changes of gene expression were evaluated after the microinjection of polyplex into living cells under conditions of nuclear factor (NF)-kappaB activation. RESULTS: Synthetic peptides with additional lysine residues were well phosphorylated by IKKbeta. Both the polymer and the polyplex were also phosphorylated by IKKbeta. The results of gel shift assay showed that the polyplex was disintegrated and free DNA was released in the presence of IKKbeta. The polyplex comprising-green fluorescent protein plasmid DNA and the polymer expressed the transgene in living cells exposed to a pro-inflammatory stimulus. CONCLUSIONS: Our concept of cell-specific gene expression was demonstrated to work in inflammatory cells. This method may provide a unique strategy for gene therapy exclusively in inflammatory cells.

Immunogenicity of hemagglutinin from A/Bar-headed Goose/Qinghai/1A/05 and A/Anhui/1/05 strains of H5N1 influenza viruses produced in Nicotiana benthamiana plants.

Highly pathogenic avian influenza (HPAI) viruses of the H5N1 subtype have been identified as a potential pandemic threat by the World Health Organization (WHO). Since 1997, these viruses have been spreading from Asia to Europe and Africa with increasing genetic and antigenic diversities. Vaccination is the preferred strategy for the prevention and control of influenza infections and the availability of a system for the rapid engineering and production of vaccines is required in the event of an influenza pandemic. In this study, we engineered and produced recombinant hemagglutinin (HA) from A/Bar-headed Goose/Qinghai/1A/05 (clade 2.2) and A/Anhui/1/2005 (clade 2.3) in Nicotiana benthamiana plants. Immunization of mice with these plant-derived HA antigens elicited serum hemagglutination inhibition (HI) and virus neutralization (VN) antibodies. These results suggest the utility of our plant-expression system for recombinant influenza vaccine production.