A simple and rapid method to monitor the disassembly and reassembly of virus-like particles.

Protein fluorescence spectra (~300-440 nm) could be used as a simple and sensitive method to monitor the disassembly and reassembly of virus-like particles (VLPs). Insect cell expressed and purified HPV-16 L1 VLPs show significantly high fluorescence intensity, whereas the fluorescence is almost quenched after disassembly by adding the reducing agent. By removing the reducing agent, the fluorescence was restored to its original intensity, indicating the reassembly of VLPs. The data are consistent with enzyme-linked immunosorbent assay (ELISA) reactivity using conformation-specific mouse monoclonal antibody. The same method could be extended to VLPs of other viruses.

E6 protein of human papillomavirus 16 (HPV16) expressed in Escherichia coli sans a stretch of hydrophobic amino acids, enables purification of GST-ΔE6 in the soluble form and retains the binding ability to p53.

Recombinant E6 expressed in Escherichia coli is known to form recalcitrant inclusion bodies even when fused to the soluble GST protein. This study describes the modification of the HPV genotype-16 oncogenic protein E6 in order to obtain it in the soluble form. The modified protein (ΔE6) was expressed in E. coli BL21 as an N-terminal fusion with GST (GST-ΔE6). ΔE6 was constructed by deleting the nucleotide sequences coding for IHDIIL (31-36 a.a), one of the highly hydrophobic peptide stretches, using splicing by overextension polymerase chain reaction (SOE-PCR). The removal of IHDIIL residues rendered the GST-ΔE6 soluble and amenable for purification involving a two step process a preliminary glutathione-GST affinity chromatography followed by gel-filtration chromatography. Evaluation of purified protein fractions by HPLC suggests that GST-ΔE6 exists as a monomer. Further, the ΔE6 in GST-ΔE6 seemed to retain the binding ability to p53 as determined by the glutathione-GST capture ELISA. Purified GST-ΔE6 we reckon, might find use as an essential reagent in immunological assays, in sero-epidemiological studies, and also in studies to delineate the structure and function of HPV16 E6.

Sequence diversity of internal transcribed spacer-1 (ITS-1) region of Eimeria infecting chicken and its relevance in species identification from Indian field samples.

Conventional method of species identification in Eimeria employs phenotypic characters of the oocysts and the site of infection in the chicken intestine, which are subjective analyses. PCR-based identification of Eimeria spp. is known to be specific and sensitive. We used internal transcribed spacer 1 (ITS-1)-based nested PCR to follow the distribution of Eimeria spp. in the field, which may be of significant value in the management of coccidiosis in chickens. In the present study, intestinal samples of chicks from commercial poultry farms, in India, suspected of having contracted Eimeria infections were analyzed using ITS-1 PCR. The PCR-amplified ITS-1 regions were also sequenced from these samples. Of 26 field samples analyzed, 19 showed the presence of multiple infections of Eimeria spp. Incidence of Eimeria tenella (80%) was found to be highest in these samples followed by Eimeria mitis (53%), Eimeria acervulina (42%), Eimeria brunetti, and Eimeria maxima (23%). Incidence of Eimeria necatrix was found to be the lowest (15%) in the samples analyzed, while none of the samples analyzed showed the presence of ITS-1 sequence from Eimeria praecox. The ITS-1 sequences amplified from Eimeria spp. in the present study showed few variations from the ITS sequences available in the GenBank database. Further studies will be required to determine whether these differences are unique to geographical locations.

Recombinant anti-hCG antibodies retained in the endoplasmic reticulum of transformed plants lack core-xylose and core-alpha(1,3)-fucose residues.

Plant-based expression systems are attractive for the large-scale production of pharmaceutical proteins. However, glycoproteins require particular attention as inherent differences in the N-glycosylation pathways of plants and mammals result in the production of glycoproteins bearing core-xylose and core-alpha(1,3)-fucose glyco-epitopes. For treatments requiring large quantities of repeatedly administered glycoproteins, the immunological properties of these non-mammalian glycans are a concern. Recombinant glycoproteins could be retained within the endoplasmic reticulum (ER) to prevent such glycan modifications occurring in the late Golgi compartment. Therefore, we analysed cPIPP, a mouse/human chimeric IgG1 antibody binding to the beta-subunit of human chorionic gonadotropin (hCG), fused to a C-terminal KDEL sequence, to investigate the efficiency of ER retrieval and the consequences in terms of N-glycosylation. The KDEL-tagged cPIPP antibody was expressed in transgenic tobacco plants or Agrobacterium-infiltrated tobacco and winter cherry leaves. N-Glycan analysis showed that the resulting plantibodies contained only high-mannose (Man)-type Man-6 to Man-9 oligosaccharides. In contrast, the cPIPP antibody lacking the KDEL sequence was found to carry complex N-glycans containing core-xylose and core-alpha(1,3)-fucose, thereby demonstrating the secretion competence of the antibody. Furthermore, fusion of KDEL to the diabody derivative of PIPP, which contains an N-glycosylation site within the heavy chain variable domain, also resulted in a molecule lacking complex glycans. The complete absence of xylose and fucose residues clearly shows that the KDEL-mediated ER retrieval of cPIPP or its diabody derivative is efficient in preventing the formation of non-mammalian complex oligosaccharides.

Typing of canine parvovirus isolates using mini-sequencing based single nucleotide polymorphism analysis.

The antigenic types of canine parvovirus (CPV) are defined based on differences in the amino acids of the major capsid protein VP2. Type specificity is conferred by a limited number of amino acid changes and in particular by few nucleotide substitutions. PCR based methods are not particularly suitable for typing circulating variants which differ in a few specific nucleotide substitutions. Assays for determining SNPs can detect efficiently nucleotide substitutions and can thus be adapted to identify CPV types. In the present study, CPV typing was performed by single nucleotide extension using the mini-sequencing technique. A mini-sequencing signature was established for all the four CPV types (CPV2, 2a, 2b and 2c) and feline panleukopenia virus. The CPV typing using the mini-sequencing reaction was performed for 13 CPV field isolates and the two vaccine strains available in our repository. All the isolates had been typed earlier by full-length sequencing of the VP2 gene. The typing results obtained from mini-sequencing matched completely with that of sequencing. Typing could be achieved with less than 100 copies of standard plasmid DNA constructs or ≤10¹ FAID50 of virus by mini-sequencing technique. The technique was also efficient for detecting multiple types in mixed infections.

Plant expressed coccidial antigens as potential vaccine candidates in protecting chicken against coccidiosis.

Coccidiosis is a disease caused by intracellular parasites belonging to the genus Eimeria. In the present study, we transiently expressed two coccidial antigens EtMIC1 and EtMIC2 as poly histidine-tagged fusion proteins in tobacco. We have evaluated the protective efficacy of plant expressed EtMIC1 as monovalent and as well as bi-valent formulation where EtMIC1 and EtMIC2 were used in combination. The protective efficacy of these formulations was evaluated using homologous challenge in chickens. We observed better serum antibody response, weight gain and reduced oocyst shedding in birds immunized with EtMIC1 and EtMIC2 as bivalent formulation compared to monovalent formulation. However, IFN-γ response was not significant in birds immunized with EtMIC1 compared to the birds immunized with EtMIC2. Our results indicate the potential use of these antigens as vaccine candidates.

Development of foot-and-mouth disease virus (FMDV) serotype O virus-like-particles (VLPs) vaccine and evaluation of its potency.

Foot-and-mouth disease (FMD) is an economically significant viral disease that rampage dairy and other livestock industries in many countries. The disease is being controlled by the use of an inactivated vaccine. However, a recombinant marker vaccine, which avoids the use of live virus, may be an option for the unambiguous differentiation of infected animals from vaccinated animals. A recombinant baculovirus clone containing P1-2A-3C coding sequences of foot-and-mouth disease virus (FMDV) serotype O(1) Manisa was generated. The FMDV structural proteins along with the 3C protease were expressed in Sf9 cells and the generation of virus like particles (VLP) was studied. The recombinant protein was formulated as vaccine using an oil adjuvant, ISA 206 and potency of the vaccine was tested in cattle. The vaccine had a potency value (PD(50)) of 5.01 and most of the vaccinated animals exhibited neutralizing antibody titers after two immunizations.

Efficacy of a recombinant chimeric anti-hCG antibody to prevent human cytotrophoblasts fusion and block progesterone synthesis.

PROBLEM: A recombinant chimeric antibody against hCG (cPIPP) has been engineered and expressed at high yield in plants. The purpose of this work was to enquire whether this antibody is competent to neutralize the bioactivity of hCG on human trophoblasts. METHODS: Cytotrophoblast cells, isolated from term placentae were maintained in culture for 3 days in presence or absence of humanized chimeric anti-hCG antibodies. Progesterone secreted was quantitated by ELISA. Fusion and cyto-architecture of the cells was studied by light and electron microscopy. Modulation of E-cadherin was investigated using RT-PCR and immunocytochemistry. RESULTS: Recombinant chimeric anti-hCG antibody blocked the synthesis of progesterone by trophoblasts. No fusion of cytotrophoblasts to form syncytium took place. E-cadherin, a vital cell adhesion molecule involved in cell-to-cell interaction did not show differentiation related decline in its expression in presence of the antibody. CONCLUSION: Recombinant chimeric anti-hCG antibody (cPIPP) was effective to neutralize hCG induced bioactivities in the human derived trophoblast cells.