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.

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.

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.

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.

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.

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.

Drug delivery system based on responses to an HIV infectious signal.

Gene therapy is a growing topic in the medical arena. Since the safety system of gene therapy has not been sufficiently established, its clinical use is limited. Recently, we developed a cell-specific gene regulation system based on a new concept, D-RECS, or Drug and Gene Delivery System Responding to Cellular Signals. We hoped here to apply this D-RECS concept to gene therapy for virus infections. In the present study, we report the design, synthesis and characterization of the functional polymers, which are able to discriminate normal and human immunodeficiency virus type 1 (HIV-1) infected cells. In the D-RECS concept, certain intracellular signals, which are extraordinary activated in the target disease cells specifically, are used as a trigger to activate a transgene expression. Thus, we paid attention to HIV protease as a target signal in this case, because HIV protease is essential for the proliferation of HIV. This protease is therefore an indicator of HIV infection. Two types of polymers were designed and synthesized using methacryloyl peptide and acrylamide with radical copolymerization as a functional gene regulator. The grafted peptide possesses a cationic protein transduction domain (PTD) sequence of HIV-Tat protein, GRKKRRQRRRPPQ for cell permeation, which are connected with polyacrylamide backbone via a consensus substrate sequence for HIV protease, SQNY/PIVQ. At first, the polymers were evaluated to see whether they possess DNA binding ability and HIV protease responsibility using gel retardation assay. The results suggested that a polymer could form a stable complex with DNA and release the DNA specifically responding to HIV protease activity. Furthermore, it was shown that this controlled release of DNA by the HIV protease signal-responsive intelligent polymer actually regulated the gene expression in the cell-free system. This system would be a useful tool for gene therapy in HIV infection, and this methodology will be applicable if the cationic peptide is replaced by another virus-specific protease, which is critical for the replication of a corresponding virus.

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.

Direct, real-time, simultaneous monitoring of intravitreal nitric oxide and oxygen in endotoxin-induced uveitis in rabbits.

Nitric oxide (NO) plays a critical role in the pathogenesis of endotoxin-induced uveitis (EIU). Since NO is a labile free radical, it is difficult to examine the dynamics of NO directly in vivo. In this study, we established a system for direct monitoring of the dynamics of NO and partial pressure of oxygen (pO(2)) in EIU in rabbits. The currents (calculated concentrations) of NO and pO(2) in the vitreous were monitored after the intravitreal injection of lipopolysaccharide (LPS). In addition, the protein concentrations and nitrite levels in the aqueous humor were analyzed. The eyes were enucleated, and a histologic study was performed on their posterior segments. The tissue slices were also immunostained with anti-nitrotyrosine as a marker of peroxinitrite and/or nitrogen-related oxidants. The NO level decreased temporarily after LPS injection and then increased from 1 to 7 h. pO(2) increased temporarily for about 30 min after LPS injection. The change in NO current was inversely proportional to pO(2) after LPS injection and vice versa. The protein concentration and nitrite level after LPS injection increased significantly. These changes were suppressed by pretreatment with N(G)-nitro-l-arginine-methyl-ester. Immunohistochemical study showed enhanced immunoreactivity of nitrotyrosine in the inflamed retina. Since nitrotyrosine was detected, it appears that NO readily reacts with oxygen to produce cytotoxic species, peroxynitrite, and/or nitrogen-related oxidants. This process may be related to the retinal injury in EIU. This monitoring system can provide useful information on dynamic changes in intravitreal NO and pO(2) for understanding EIU.

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.

The features and shortcomings for gene delivery of current non-viral carriers.

Since the viral vector for gene therapy has serious problems, including oncogenesity and other adverse effects, non-viral carriers have attracted a great deal of attention. Non-viral carriers are expected to achieve gene therapy without serious side effects. However, the most critical issue of gene delivery by non-viral carriers is the low-expression efficiencies of the desired gene. In order to apply non-viral carriers for gene therapy in practical clinical usage, further understanding of the cellular barriers against gene delivery is a prerequisite. Moreover, additional intelligent concepts for gene delivery are also needed. We will summarize the features and shortcomings of currently developed non-viral delivery systems. Especially, we will address the current progress of cationic lipids (lipoplex) and cationic polymers (polyplex) in terms of transfection efficiency. Furthermore, our group has developed a system that responds to the particular intracellular signals of target disease cells. We have named this gene delivery system a drug delivery system based on responses cellular signal (D-RECS). We will introduce this new concept of intelligent non-viral delivery system that our group recently developed.

A highly attenuated rabies virus HEP-Flury strain reverts to virulent by single amino acid substitution to arginine at position 333 in glycoprotein.

An amino acid at position 333 in the glycoprotein of several fixed rabies virus strains is responsible for the pathogenicity in adult mice. Substitution of arginine at this position largely reduces the viral pathogenicity in adult mice. Attenuation by this single amino acid substitution has been established by using escape mutants selected by monoclonal antibodies and point-mutated virus generated by reverse-genetics. A highly attenuated HEP-Flury strain, which was selected by serial passages in cell cultures, has glutamine at this position. In this study, a point-mutated rHEP333R virus, having arginine at position 333, was generated and examined for the responsibility of this substitution in rabies pathogenicity. The rHEP333R acquired an ability to spread and propagate in mouse brain but the parental rHEP did not. The pathogenicity of rHEP333R to adult mice by intracerebral inoculation largely increased. We confirmed that an arginine at position 333 contributed to reversion of the pathogenicity in a highly attenuated HEP-Flury strain.

Genetic analysis of dog rabies viruses circulating in Bangkok.

The genetic diversity of the rabies virus glycoprotein (G) gene isolated from individual rabid dogs (inter-hosts) and within a single infected dog (intra-host) has been analyzed in an effort to better understand selective pressures and population shifts among rabies viruses circulating in Bangkok. Comparison of individual master sequences among inter-hosts revealed that the dog virus isolates circulating in Bangkok were phylogenetically closely related. The ectodomain of the glycoprotein was highly conserved among the virus isolates. Furthermore, the genetic diversity of the G gene within an intra-host was assessed by comparing the cloned sequences in the virus population. The comparisons revealed that rabies virus circulating in an intra-host consisted of closely related heterogenous populations with minor substitutions at nucleotide (0.19%) and amino acid levels.

Anti-stress activity of mulberry juice in mice.

The possible anti-stress activity of mulberry juice was investigated in mice. When mice were subjected to water immersion restraint stress at 25 degrees C for 8 h, the plasma lipid peroxide level, determined by the d-ROMs test performed 12 h thereafter, was almost doubled. After administration of mulberry juice one or two weeks before the stress loading, the lipid peroxidation was completely blocked. Administration of mulberry juice after the stress loading, without pre-administration, was also protective. ESR spectroscopy revealed that mulberry juice scavenged superoxide anion (generated by hypoxanthine and xanthine oxidase reaction), hydroxyl radical (produced by the Fenton reaction) and NO radical (generated by a NO donor) at approximately 50% efficiency of blueberry juice. Mulberry juice produced smaller amounts of radical at neutral to alkaline pH. The cytotoxic and anti-HIV activities of mulberry juice were 18% and >4-fold those of blueberry juice, respectively. These data suggest that the anti-stress activity of mulberry juice in vivo may be derived from its radical scavenging activity.

Glucose-lowering effect of powder formulation of African black tea extract in KK-A(y)/TaJcl diabetic mouse.

We observed the suppressive effect of a powder formulation of African black tea extract prepared from the leaves of Camellia sinensis on type 2 non-insulin dependent diabetic mice, KK-A(y)/TaJcl. Black tea extract significantly showed suppressive effect of the elevation of blood glucose on oral glucose tolerance test of 8 week-old KK-A(y)/TaJcl mice (P < 0.05). Long-term treatment with black tea extract showed significant suppression of post-prandial blood glucose and obesity (P < 0.05). The weight of the intestine of mice treated with black tea extract was significantly reduced (P < 0.05). From these results, African black tea used in this study showed a suppressive effect on the elevation of blood glucose during food intake and the body weight.

A novel rapid fluorescent focus inhibition test for rabies virus using a recombinant rabies virus visualizing a green fluorescent protein.

Virus-neutralizing antibodies (VNAs) against rabies virus play a major role in protection from rabies. The rapid fluorescent focus inhibition test (RFFIT) has been internationally recognized as a standard in vitro test for measuring the VNA. CVS strain of rabies virus is used as challenge virus and the infected cells are indirectly detected by staining with fluorescein isothiocyanate (FITC)-conjugated rabies antibody which is expensive and high-quality products are often in short supply. In this study, a recombinant rabies virus strain carrying a green fluorescent protein (GFP) gene, rHEP-GFP, was used as a challenge virus in the virus neutralization assay. Expression of the GFP could be readily detected in the infected cells under a fluorescent microscope. This novel RFFIT modification RFFIT-GFP is a neutralization test, and it is based on the sound principle of the standard RFFIT using 96-well plates. VNA titers in 25 human, 18 canine and 15 horse sera have been compared between the RFFIT and RFFIT-GFP methods. The results obtained by the both methods showed good agreement between both methods in all sera investigated (coefficient of correlation, r = 0.98). It allowed direct detection of virus by expression of GFP and might be applicable for other viruses. The novel method is convenient, economical and a reliable tool not requiring expensive FITC-conjugated antibody for routine rabies VNA assays.

Cytotoxic activity of azulenequinones against human oral tumor cell lines.

We investigated twenty-seven azulenequinone derivatives for their relative cytotoxicity against three human normal cell lines (HGF, HPC, HPLF) and four human tumor cell lines (HSG, HSC-2, HSC-3, HL-60). Parent 1,5-azulenequinone showed potent and some tumor-specific cytotoxicity. Halogenated derivatives of 1,5- and 1,7-azulenequinone showed potent cytotoxicity, but lower tumor-specific cytotoxicity. In contrast to other azulenequinones, amino derivatives such as 3-amino-1,5- and 1, 7-azulenequinones showed relatively lower cytotoxic activity. The 3-Phenoxy-1,5-azuleneqinone derivative showed higher cytotoxicity than the 3-phenoxy-1, 7-azulenequinone derivative. 1,5- and 1,7-Azulenequinones generally showed higher cytotoxicity, as compared with tropolones and azulene derivatives. 3- (3-Guaiazulenyl)-1, 5-azulenequinone [12] and 7-isopropyl-3- (4-methylanilino)-2-methyl- 1, 5-azulenequinone [24] showed a relatively higher TS value and induced apoptosis (internucleosomal DNA fragmentation, activation of caspases 3, 8 and 9) in HL-60 and HSC-2 cells, possibly via the activation of both mitochondria-independent (extrinsic) and -dependent (intrinsic) pathways. Western blot analysis showed that [24] slightly increased the intracellular concentration of pro-apoptotic proteins (Bad, Bax) in HSC-2 cells, whereas [12] was much less active. None of the twenty-seven azulenequinones showed anti-HIV activity. These results suggest [12] and [24] as possible candidates for future cancer chemotherapy.

Cytotoxicity and radical modulating activity of Moxa smoke.

The biological activities of Moxa, used as moxibustion, have not been well documented. We investigated here Moxa smoke for its tumor-specific cytotoxicity, anti-HIV activity, radical intensity and radical scavenging activity, in comparison with previously published data of Moxa extract. Moxa smoke showed slightly higher cytotoxicity against human tumor cell lines (oral squamous cell carcinoma HSC-2, HSC-3, promyelocytic leukemia HL-60) than against normal oral cells (gingival fibroblast HGF, pulp cell HPC, periodontal ligament fibroblast HPLF), yielding a tumor specificity index of 1.29. Moxa smoke dose-dependently induced internucleosomal DNA fragmentation, activation of caspases 3, 8 and 9, and slightly modified the expression of apoptosis-related proteins (Bcl-2, Bad, Bax) in HL-60 cells, but to much lesser extents than attained by positive controls (UV irradiation, actinomycin D treatment). ESR spectroscopy showed that Moxa smoke generated semiquinone-type radicals under alkaline conditions, and scavenged O2(-), hydroxyl radical, singlet oxygen and NO. All Moxa smoke preparations showed no apparent anti-HIV activity. These data demonstrate the antitumor potential of Moxa smoke.