Silkworm expression system as a platform technology in life science

Many recombinant proteins have been successfully produced in silkworm larvae or pupae and used for academic and industrial purposes. Several recombinant proteins produced by silkworms have already been commercialized. However, construction of a recombinant baculovirus containing a gene of interest requires tedious and troublesome steps and takes a long time (3–6 months). The recent development of a bacmid, Escherichia coli and Bombyx mori shuttle vector, has eliminated the conventional tedious procedures required to identify and isolate recombinant viruses. Several technical improvements, including a cysteine protease or chitinase deletion bacmid and chaperone-assisted expression and coexpression, have led to significantly increased protein yields and reduced costs for large-scale production. Terminal N-acetyl glucosamine and galactose residues were found in the N-glycan structures produced by silkworms, which are different from those generated by insect cells. Genomic elucidation of silkworm has opened a new chapter in utilization of silkworm. Transgenic silkworm technology provides a stable production of recombinant protein. Baculovirus surface display expression is one of the low-cost approaches toward silkworm larvae-derived recombinant subunit vaccines. The expression of pharmaceutically relevant proteins, including cell/viral surface proteins, membrane proteins, and guanine nucleotide-binding protein (G protein) coupled receptors, using silkworm larvae or cocoons has become very attractive. Silkworm biotechnology is an innovative and easy approach to achieve high protein expression levels and is a very promising platform technology in the field of life science. Like the “Silkroad,” we expect that the “Bioroad” from Asia to Europe will be established by the silkworm expression system

Preserved antigenicity of HIV-1 p24 produced and purified in high yields from plants inoculated with a tobacco mosaic virus (TMV)-derived vector

Production of structural proteins from foot-and-mouth disease virus (FMDV) and bovine herpes virus (BHV-1) in Nicotiana benthamiana through the use of a tobacco mosaic virus-based vector (TMV-30B) has been reported previously. The development of the TMV-30B-HISc vector, a new version that adds a C-terminal histidine (His) sequence to the foreign protein expressed is described. Coding sequences from the FMDV VPl protein and the core protein, p24, from a clade C HIV-1 isolate from Zambia were cloned into the new vector and infective RNAs were generated for each construct to inoculate N. benthamiana plants. His-tagged proteins were purified from inoculated leaves using immobilized metal affinity chromatography (IMAC) as detected by Coomassie blue staining and proteins were further characterized in Western blot assays using a commercial anti-6xHis mAb and specific polyclonal antisera for each protein. While yields obtained for the VPl-His protein after purification were similar to those in crude extracts obtained with the previous TMV-VPl vector, p24-His yields were 10–15 times higher than those of VPl-His. Twenty-five grams of TMV-p24-HISc inoculated leaves were processed to obtain 2.5 mg of isolated p24-His and the recombinant protein was inoculated in rabbits to test immunogenicity and antigenic integrity of the plant-produced p24-His. Animals developed a strong and specific humoral response to the p24-His after the first booster and immune sera was able to recognize the native p24 from a different clade expressed on the surface of the HIV-1 chronically infected HUT78/ARV T-cell line. Importantly, the recombinant p24-His proved its efficiency by confirming the serology of 117 samples previously tested by two rapid HIV-1 tests, thus representing an excellent alternative for production of highly specific diagnostic reagents for HIV endemic regions in the developing world

Multimerization of peptide antigens for production of stable immunogens in transgenic plants

Previous literature addressing the production of recombinant proteins in heterologous systems has consistently shown that proteins capable of forming complex structures tend to accumulate within host cells at relatively higher levels than monomeric forms. In this report, we translationally fused a 21-aminoacids long highly immunogenic peptide (2L21), derived from canine parvovirus (CPV) VP2 protein to a 41-aminoacid long tetramerization domain (TD) from the transcriptional factor p53. The chimerical DNA construction 2L21-TD was cloned in a binary plant transformation vector and used to transform Arabidopsis thaliana plants. Fifteen of the 25 transgenic lines obtained in the experiment showed detectable 2L21-TD RNA accumulation and from these we chose 4 to study 2L21-TD protein accumulation. Non-denaturing immunoblotting assays revealed that 2L21-TD chimeras effectively formed tetrameric complexes with yields reaching up to 12 μg/mg of soluble protein. Mice immunized by oral or intraperitoneal routes with crude protein extracts containing 2L21-TD protein were able to detect both 2L21-synthetic peptide and CPV VP2 proteins, with titers similar to those elicited by a previously reported fusion between 2L21 and the β-glucuronidase protein. These results demonstrate that multimerization directed by the small TD domain contributed to the stabilization and consequently to the accumulation of the 2L21 peptide in transgenic plants, without altering its native antigenicity and immunogenicity. © 2006 Elsevier B.V. All rights reserved

Early Adaptive Immune Responses in the Respiratory Tract of Foot and- Mouth Disease Virus-Infected Cattle

Foot-and-mouth disease (FMD) is a highly contagious viral disease which affects both domestic and wild biungulate species. This acute disease, caused by the FMD virus (FMDV), usually includes an active replication phase in the respiratory tract for up to 72 h postinfection, followed by hematogenous dissemination and vesicular lesions at oral and foot epithelia. The role of the early local adaptive immunity of the host in the outcome of the infection is not well understood. Here we report the kinetics of appearance of FMDV-specific antibody-secreting cells (ASC) in lymphoid organs along the respiratory tract and the spleen in cattle infected by aerosol exposure. While no responses were observed for up to 3 days postinfection (dpi), all animals developed FMDV-ASC in all the lymphoid organs studied at 4 dpi. Tracheobronchial lymph nodes were the most reactive organs at this time, and IgM was the predominant isotype, followed by IgG1. Numbers of FMDV-ASC were further augmented at 5 and 6 dpi, with an increasing prevalence in upper respiratory organs. Systemic antibody responses were slightly delayed compared with the local reaction. Also, IgM was the dominant isotype in serum at 5 dpi, coinciding with a sharp decrease of viral RNA detection in peripheral blood. These results indicate that following aerogenous administration, cattle develop a rapid and vigorous genuine local antibody response throughout the respiratory tract. Time course and isotype profiles indicate the presence of an efficient T cell-independent antibody response which drives the IgM-mediated virus clearance in cattle infected by FMDV aerosol exposure

Induction of anti foot and mouth disease virus T and B cell responses in cattle immunized with a peptide representing ten amino acids of VP1

We previously demonstrated that the immunization of cattle with a synthetic peptide representing the amino acid sequence of foot and mouth disease virus (FMDV) type O1 Campos VP1 residues 135-160 (p135-160), containing immunodominant T and B epitopes, was able to induce a strong neutralizing antibody (NA) response. The epitope mapping of p135-160 identified T and B epitopes in the area restricted to amino acid residues 135-144 (Zamorano et al. 1994, Virology 201; 1995, Virology 212). We are now reporting that, although immunization with a synthetic peptide covering amino acids 135-144 (p135-144) failed to elicit an anti-FMDV response, a synthetic peptide representing a tandem duplication of the VP1 epitope 135-144 (p135-144 x 2) was very efficient in inducing a strong NA response in cattle. Both the antibody and T cell responses elicited by p135-144 x 2 were highly specific for the VP1 135-144 sequence since no reactivity was detected against synthetic peptides representing the 140-160 sequence of VP1. Additionally, both responses to B and T epitopes were long lasting in the immunized cattle. These results constitute a good example of the improvement of the immune response by rational handling of precisely identified B and T epitopes. To our knowledge, this is the shortest native amino acid sequence to induce a significant NA response to FMDV in cattle

Bovine herpes virus gD protein produced in plants using a recombinant tobacco mosaic virus (TMV) vector possesses authentic antigenicity

A tobacco mosaic virus (TMV)-based vector was utilized for expression of a cytosolic form of the bovine herpesvirus type 1 (BHV-1) protein glycoprotein D (gDc). Nicotiana benthamiana plants were harvested 7 days after inoculation with RNA transcripts derived from the TMV-gDc recombinant virus. Recombinant gDc protein of expected electrophoretic mobility accumulated in inoculated leaves to a concentration of about 20 μg/g of fresh leaf tissue. Oil-based vaccines were formulated with crude foliar extracts to immunize mice parentally. After a single injection, animals developed a sustained and specific response to both the isolated gD and native virus particles. Cattle vaccinated with the same gDc containing extracts developed specific humoral and cellular immune responses directed against both the viral gD and BHV-1 particles. Most importantly, animals vaccinated with the plant-produced gDc showed good levels of protection after challenge with the virulent BHV-1. Virus excretion was drastically reduced in these animals, reaching levels comparable to animals vaccinated with a commercial BHV-1 vaccine. The positive immunological characterization obtained for the gDc, indicated that an important part of the natural conformation was retained in the plant recombinant protein

DIVA diagnostic of Aujeszky's disease using an insect-derived virus glycoprotein E

Commercial vaccines against Aujeszky's disease are mainly formulated using deleted versions of attenuated or inactivated Pseudorabies virus (PRV) particles lacking of the structural glycoprotein E (gE). Complementary diagnostic assays used to differentiate infected from vaccinated animals (DIVAs), are based on the detection of serum antibodies against gE. A recombinant version of the PRV gE protein was expressed in a baculovirus vector system in Trichoplusia ni insect larvae in order to obtain this diagnostic reagent for large scale diagnosis at reduced costs. A recombinant gE gene (gEr), lacking of signal peptide and transmembrane domains, was cloned into a modified baculovirus vector to allow glycosylation of the protein and its subsequent exportation to the extracellular space. Analysis by SDS-PAGE, Western-blotting and glycoprotein staining revealed that a glycosylated protein of the expected electrophoretic mobility was obtained in infected larvae. Time course experiments revealed that maximum expression levels were reached 72 h post-infection using 104 pfu of the recombinant baculovirus (BACgEr) per inoculated larva. An indirect PRV gE-ELISA was developed using gEr as a coating antigen. A comparison between larvae-derived PRV gE-ELISA and two commercially available PRV diagnostic kits showed good correlation between assays and better sensitivity when testing certain sera pig samples using the gEr ELISA. More than 30,000 ELISA determinations could be performed from crude extracts obtained from a single larva infected with the recombinant baculovirus, indicating the feasibility of this strategy for inexpensive production of glycosylated antigens for PRV diagnosis. © 2008 Elsevier B.V. All rights reserved

Development of a low-cost, insect larvae-derived recombinant subunit vaccine against RHDV

Vaccine antigens against rabbit hemorrhagic disease virus (RHDV) are currently derived from inactivated RHDV obtained from livers of experimentally infected rabbits. Several RHDV-derived recombinant immunogens have been reported. However, their application in vaccines has been restricted due to their high production costs. In this paper, we describe the development of an inexpensive, safe, stable vaccine antigen for RHDV. A baculovirus expressing a recombinant RHDV capsid protein (VP60r) was used to infect Trichoplusia ni insect larvae. It reached an expression efficiency of 12.5% of total soluble protein, i.e. ∼ 2 mg of VP60r per larva. Preservation of the antigenicity and immunogenicity of the VP60r was confirmed by immunological and immunization experiments. Lyophilized crude larvae extracts, containing VP60r, were stable, at room temperature, for at least 800 days. In all cases, rabbits immunized with a single dose of VP60r by the intramuscular route were protected against RHDV challenge. Doses used were as low as 2 μg of VP60r in the presence of adjuvant or 100 μg without one. Orally administered VP60r in the absence of an adjuvant gave no protection. The potential costs of an RHDV vaccine made using this technology would be reduced considerably compared with producing the same protein in insect cells maintained by fermentation. In conclusion, the larva expression system may provide a broad-based strategy for production of recombinant subunit antigens (insectigens) for human or animal medicines, especially when production costs restrain their use. © 2007 Elsevier Inc. All rights reserved

Optimization and Validation of Recombinant Serological Tests for African Swine Fever Diagnosis Based on Detection of the p30 Protein Produced in Trichoplusia ni Larvae

We describe the validation of an enzyme-linked immunosorbent assay (ELISA) and confirmatory immunoblotting assays based on a recombinant p30 protein (p30r) produced in insect larvae using a baculovirus vector. Such validation included the following: (i) the scaling up and standardization of p30r production and the associated immunoassays, (ii) a broad immunological analysis using a large number of samples (a total of 672) from Spain and different African locations, and (iii) the detection of the ASF virus (ASFV)-antibody responses at different times after experimental infection. Yields of p30r reached up to 15% of the total protein recovered from the infected larvae at 3 days postinfection. Serological analysis of samples collected in Spain revealed that the p30r-based ELISA presented similar sensitivity to and higher specificity than the conventional Office International des Epizooties-approved ASFV ELISA. Moreover, the p30r ELISA was more sensitive than the conventional ELISA test in detecting ASFV-specific antibodies in experimentally infected animals at early times postinfection. Both the recombinant and conventional ELISAs presented variable rates of sensitivity and specificity with African samples, apparently related to their geographical origin. Comparative analyses performed on the sequences, predicted structures, and antigenicities of p30 proteins from different Spanish and African isolates suggested that variability among isolates might correlate with changes in antigenicity, thus affecting detection by the p30r ELISA. Our estimations indicate that more than 40,000 ELISA determinations and 2,000 confirmatory immunoblotting tests can be performed with the p30r protein obtained from a single infected larva, making this a feasible and inexpensive strategy for production of serological tests with application in developing countries