Induction of potent Th1-type immune responses from a novel DNA vaccine for West Nile virus New York isolate (WNV-NY1999).

West Nile virus (WNV) is a vectorborne pathogen that induces brain inflammation and death. Recently, confirmed cases of infection and deaths have occurred in the United States Mid-Atlantic region. In this study, a DNA vaccine encoding the WNV capsid protein was constructed, and the in vivo immune responses generated were investigated in DNA vaccine-immunized mice. Antigen-specific humoral and cellular immune responses were observed, including a potent induction of antigen-specific Th1 and cytotoxic T lymphocyte responses. Strong induction of Th1-type immune responses included high levels of antigen-specific elaboration of the Th1-type cytokines interferon-gamma and interleukin-2 and beta-chemokines RANTES (regulated upon activation, normal T cell-expressed and secreted) and macrophage inflammatory protein-1beta. Dramatic infiltration of CD4 and CD8 T cells and macrophages also was observed at the muscle injection site. These results support the potential utility of this method as a tool for developing immunization strategies for WNV and other emerging pathogens.

Choice of expression vector alters the localization of a human cellular protein.

The fusion of synthetic epitopes with proteins of interest is an important tool in the identification and characterization of recombinant proteins. Several mammalian expression vectors are commercially available containing unique identification tags or epitopes. These vectors offer a great advantage to researchers, as highly specific antibodies and purification resins against these specific epitopes are readily available. The tags facilitate immunologic assays and the purification of the recombinant proteins. The fusion of these epitopes with the recombinant proteins is not expected to alter the behavior of the protein of interest. In this report, we demonstrate that the mere expression of a cellular protein, hVIP/mov34, which we earlier identified as a cellular HIV-1 Vpr ligand, in two different vectors clearly altered its localization pattern in HeLa cells. Specifically, cloning of hVIP/mov34 in pcDNA3/HisA resulted in its nuclear localization, whereas the expression of this gene from a TOPO cloning expression vector, pcDNA3.1/V5/His, resulted in cytoplasmic expression. The native staining pattern of hVIP/mov34 using polyclonal antisera raised against hVIP/mov34 demonstrated cytoplasmic staining. During cloning, other leader sequences intended for targeting this protein into a cytoplasmic or a nuclear location were not fused to the actual ORF of this protein. Also, the amino acid sequence of the fusion region arising from cloning of hVIP/mov34 in both vectors does not match any reported NLS sequences. These results indicate that the choice of the expression vectors, as well as the position of synthetic epitopes, can significantly alter the behavior and the biology of recombinant proteins. This result suggests the need for a careful examination of these features when characterizing a newly identified protein.

Immunogenicity of a novel DNA vaccine cassette expressing multiple human immunodeficiency virus (HIV-1) accessory genes.

OBJECTIVE: To develop an HIV-1 accessory gene immunogen using a DNA vaccine approach. METHODS: HIV-1 accessory genes vif, vpu and nef were modified to express under the control of a single promoter with cellular proteolytic cleavage sites between the coding sequences (VVN-P). Immune responses induced by these constructs were evaluated in mice. RESULTS: DNA vaccine construct (pVVN-P) expressing Vif, Vpu and Nef was processed and the fusion protein was cleaved appropriately. Vif, Vpu and Nef as a fusion protein with proteolytic cleavage sites (VVN-P) is able to induce a significant level of cellular immune responses. We also observed that accessory genes Vif, Vpu and Nef (VVN-P) induced an effective T helper 1 proliferative response measured by cytokine production. Furthermore, expression cassette pVVN-P was able to induce cytotoxic T lymphocyte (CTL) responses against diverse HIV-1 viruses in infected target cells. CONCLUSION: We conclude that cell-mediated immune responses induced by accessory gene constructs from clade B may have a broader recognition of divergent HIV-1 viruses and should be further examined for both prophylactic and therapeutic vaccination schemes against HIV-1.

Complete mineralization of methylparathion by Pseudomonas sp. A3.

Organophosphorus insecticides are widely used in agriculture. Despite their biodegradable nature, some are highly toxic and their residues are found in the environment. Reports on the mineralization of a spectrum of these insecticides by a single potential strain are scarce. We have isolated a soil isolate, Pseudomonas sp. A3, through enrichment technique, able to degrade methylparathion (MP), malathion, monocrotophos, and Diazinon. The potential of this strain to mineralize MP as a carbon and/or phosphorus source has been evaluated. On hydrolysis of MP, the aromatic portion (p-nitrophenol) was used as a carbon and energy source whereas the alkyl moiety (dithiomethylphosphorothioate) was broken down for the phosphorus source. The results from the experiments involving [U-14C]p-nitrophenol provided the evidence for incorporation of carbon into the cellular constituents and release of CO2 from this insecticide. During the breakdown of MP, nitrite was released as a catabolic by-product.

Short communication: Methylparathion degradation by Pseudomonas sp. A3 immobilized in sodium alginate beads.

An organophosphate-degrading soil isolate of Pseudomonas sp. A3, immobilized at 5% (wet wt/v) cell mass in 3% (w/v) sodium alginate beads, detoxified 99% of 1 mM methylparathion in 48 h. The beads were re-usable for five batches, the sixth batch only giving 73% methylparathion removal.