Correspondence between the T-cell epitope responses of vaccine immunogens and those of pathogen antigens is critical to vaccine efficacy. In the present study, we analyzed the spectrum of immune responses of mice to three different forms of the SARS coronavirus nucleocapsid (N): (1) exogenous recombinant protein (N-GST) with Freund's adjuvant; (2) DNA encoding unmodified N as an endogenous cytoplasmic protein (pN); and (3) DNA encoding N as a LAMP-1 chimera targeted to the lysosomal MHC II compartment (p-LAMP-N). Lysosomal trafficking of the LAMP/N chimera in transfected cells was documented by both confocal and immunoelectron microscopy. The responses of the immunized mice differed markedly. The strongest T-cell IFN-gamma and CTL responses were to the LAMP-N chimera followed by the pN immunogen. In contrast, N-GST elicited strong T cell IL-4 but minimal IFN-gamma responses and a much greater antibody response. Despite these differences, however, the immunodominant T-cell ELISpot responses to each of the three immunogens were elicited by the same N peptides, with the greatest responses being generated by a cluster of five overlapping peptides, N76-114, each of which contained nonameric H2d binding domains with high binding scores for both class I and, except for N76-93, class II alleles. These results demonstrate that processing and presentation of N, whether exogenously or endogenously derived, resulted in common immunodominant epitopes, supporting the usefulness of modified antigen delivery and trafficking forms and, in particular, LAMP chimeras as vaccine candidates. Nevertheless, the profiles of T-cell responses were distinctly different. The pronounced Th-2 and humoral response to N protein plus adjuvant are in contrast to the balanced IFN-gamma and IL-4 responses and strong memory CTL responses to the LAMP-N chimera.

Domoic acid (DA), an agonist of non-N-methyl-D-aspartate (non-NMDA) receptor subtype including kainate receptor, was identified as a potent neurotoxin showing involvement in neuropathological processes like neuronal degeneration and atrophy. In the past decade evidence indicating a role for excitatory amino acids in association with neurological disorders has been accumulating. Although the mechanisms underlying the neuronal damage induced by DA are not yet fully understood, many intracellular processes are thought to contribute towards DA-induced excitotoxic injury, acting in combination leading to cell death. In this review article, we report the leading hypotheses in the understanding of DA-induced neurotoxicity, which focus on the role of DA in neuropathological manifestations, the formation of the retrograde messenger molecule nitric oxide (NO) for the production of free radicals in the development of neuronal damage, the activation of glial cells (microglia and astrocytes) in response to DA-induced neuronal damage and the neuroprotective role of melatonin as a free radical scavenger or antioxidant in DA-induced neurotoxicity. The possible implications of molecular mechanism underlying the neurotoxicity in association with necrosis, apoptosis, nitric oxide synthases (nNos and iNOS) and glutamate receptors (NMDAR1 and GluR2) related genes and their expression in DA-induced neuronal damage in the hippocampus have been discussed.

A genetic vaccine for West Nile virus (WN) has been synthesized with the WN premembrane-envelope (WN preM-E) gene sequences encoded as a chimera with the transmembrane and carboxyl terminal domains of the lysosome-associated membrane protein (LAMP). The LAMP sequences are used to direct the antigen protein to the major histocompatibility class II (MHC II) vesicular compartment of transfected professional antigen-presenting cells (APCs). Vaccine constructs encoding the native WN preM-E and WN preM-E/LAMP chimera were synthesized in pVAX1 and pITR plasmid backbones. Extracts of human fibroblast 293 and monkey kidney COS-7 cells transfected with the WN preM-E/LAMP chimera constructs contained much greater amounts of E than did the cells transfected with constructs encoding the native WN preM-E. This difference in the concentration of native E and the E/LAMP chimera in transfected cells is attributed to the secretion of native E. The amount of preM protein in cell extracts, in contrast to the E protein, and the levels of DNA and RNA transcripts, did not differ between WN preM-E- and WN preM-E/LAMP-transfected cells. Additionally, confocal and immunoelectron microscopic analyses of transfected B cells showed localization of the WN preM-E/LAMP chimera in vesicular compartments containing endogenous LAMP, MHC II, and H2-M, whereas native viral preM-E lacking the LAMP sequences was distributed within the cellular vesicular network with little LAMP or MHC II association. Mice immunized with a DNA construct expressing the WN preM-E/LAMP antigen induced significant antibody and long-term neutralization titers in contrast to the minimal and short-lived neutralization titer of mice vaccinated with a plasmid expressing the untargeted antigen. These results underscore the utility of LAMP targeting of the WN envelope to the MHC II compartments in the design of a genetic WN vaccine.

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