Preparation and immunogenic properties of a recombinant West Nile subunit vaccine.

While several West Nile vaccines are being developed, none are yet available for humans. In this study aimed at developing a vaccine for humans, West Nile virus (WNV) envelope protein (E) and non-structural protein 1 (NS1) were produced in the Drosophila S2 cell expression system. The C-terminal 20% of the E protein, which contains the membrane anchor portion, was deleted, thus allowing for efficient secretion of the truncated protein (80E) into the cell culture medium. The proteins were purified by immunoaffinity chromatography (IAC) using monoclonal antibodies that were flavivirus envelope protein group specific (for the 80E) or flavivirus NS1 group specific (for NS1). The purified proteins were produced in high yield and used in conjunction with adjuvant formulations to vaccinate mice. The mice were tested for both humoral and cellular immune responses by a plaque reduction neutralization test and ELISA, and by lymphocyte proliferation and cytokine production assays, respectively. The results revealed that the 80E and the NS1 proteins induced both high-titered ELISA and neutralizing antibodies in mice. Splenocytes from immunized mice, cultured in vitro with the vaccine antigens as stimulants, showed excellent proliferation and production of cytokines (IFN-gamma, IL-4, IL-5, and IL-10). The level of antigen-stimulated lymphocyte proliferation and cytokine production was comparable to the level obtained from mitogen (phytohemagglutinin or pokeweed) stimulation, indicating a robust cellular response as well. These findings are encouraging and warrant further in vivo studies to determine the protective efficacy of the WNV vaccine candidate.

Definition of the M-conotoxin superfamily: characterization of novel peptides from molluscivorous Conus venoms.

Most of the >50,000 different pharmacologically active peptides in Conus venoms belong to a small number of gene superfamilies. In this work, the M-conotoxin superfamily is defined using both biochemical and molecular criteria. Novel excitatory peptides purified from the venoms of the molluscivorous species Conus textile and Conus marmoreus all have a characteristic pattern of Cys residues previously found in the mu-, kappaM-, and psi-conotoxins (CC-C-C-CC). The new peptides are smaller (12-19 amino acids) than the mu-, kappaM-, and psi-conotoxins (22-24 amino acids). One peptide, mr3a, was chemically synthesized in a biologically active form. Analysis of the disulfide bridges of a natural peptide tx3c from C. textile and synthetic peptide mr3a from C. marmoreus showed a novel pattern of disulfide connectivity, different from that previously established for the mu- and psi-conotoxins. Thus, these peptides belong to a new group of structurally and pharmacologically distinct conotoxins that are particularly prominent in the venoms of mollusc-hunting Conus species. Analysis of cDNA clones encoding the novel peptides as well as those encoding mu-, kappaM-, and psi-conotoxins revealed highly conserved amino acid residues in the precursor sequences; this conservation in both amino acid sequence and in the Cys pattern defines a gene superfamily, designated the M-conotoxin superfamily. The peptides characterized can be provisionally assigned to four distinct groups within the M-superfamily based on sequence similarity within and divergence between each group. A notable feature of the superfamily is that two distinct structural frameworks have been generated by changing the disulfide connectivity on an otherwise conserved Cys pattern.

A simple method for the rapid purification of copia virus-like particles from Drosophila Schneider 2 cells.

Drosophila retrotransposon copia produces virus-like particles (VLPs) in cultured Drosophila cells. The VLPs contain copia RNA and reverse transcriptase activity, and thus, play a major role in copia replication. Here, we report a rapid and simple method for the purification of copia VLPs from Drosophila Schneider 2 (S2) cells. The VLP purification procedure consists of a series of short centrifugation steps and eliminates the tedious and time-consuming performance of sucrose, metrizamide and cesium chloride (CsCl) gradients. The purity and presence of VLPs at different purification steps was monitored by transmission electron microscopy (TEM), immunochemical detection methods, and by Northern blot analysis. In addition to providing a fast protocol for VLP purification, our results also show that copia particles are mainly located in the nucleus of S2 cells. This new protocol may find broad applications for the purification of various other VLPs, and thus, may be of great value to other investigators with similar interests.