A tris (2-carboxyethyl) phosphine (TCEP) related cleavage on cysteine-containing proteins.

Introduced in the late 1980s as a reducing reagent, Tris (2-carboxyethyl) phosphine (TCEP) has now become one of the most widely used protein reductants. To date, only a few studies on its side reactions have been published. We report the observation of a side reaction that cleaves protein backbones under mild conditions by fracturing the cysteine residues, thus generating heterogeneous peptides containing different moieties from the fractured cysteine. The peptide products were analyzed by high performance liquid chromatography and tandem mass spectrometry (LC/MS/MS). Peptides with a primary amine and a carboxylic acid as termini were observed, and others were found to contain amidated or formamidated carboxy termini, or formylated or glyoxylic amino termini. Formamidation of the carboxy terminus and the formation of glyoxylic amino terminus were unexpected reactions since both involve breaking of carbon-carbon bonds in cysteine.

Murine cytomegalovirus abortively infects human dendritic cells, leading to expression and presentation of virally vectored genes.

Dendritic cells (DC) are potent antigen-presenting cells that play a crucial role in antigen-specific immune responses. Thus, the targeting of exogenous antigens to DC has become a popular approach for cancer immunotherapy and vaccine development. In this report, we studied the interplay between murine cytomegalovirus (MCMV) and human monocyte-derived DC. The results showed that an enhanced green fluorescent protein (EGFP)-encoding, replication-competent MCMV vector underwent abortive infection in human DC; this was accompanied by the efficient expression of EGFP. Infection of human DC by this vector resulted in a modest increase in the expression of cell surface proteins associated with DC maturation and has no significant effect on the immunostimulatory function of the cells, as reflected by their ability to support T-cell proliferation in a mixed-lymphocyte reaction. Finally, an MCMV vector encoding the human immunodeficiency virus type 1 (HIV-1) gp120 envelope glycoprotein was constructed and used to infect cultured human DC. The infected DC were shown to be capable of stimulating the expansion of autologous, gp120-specific, class I-restricted T lymphocytes from an HIV-1-negative donor, as determined by tetramer staining and enzyme-linked immunospot analysis. Taken together, these results suggest that MCMV may have potential utility as a vector for human vaccine development.

Expression of human immunodeficiency virus type 1 gp120 from herpes simplex virus type 1-derived amplicons results in potent, specific, and durable cellular and humoral immune responses.

Herpes simplex virus type 1 (HSV-1) infects a wide range of cells, including dendritic cells. Consequently, HSV-1 vectors may be capable of eliciting strong immune responses to vectored antigens. To test this hypothesis, an HSV-1 amplicon plasmid encoding human immunodeficiency virus type 1 gp120 was constructed, and murine immune responses to helper virus-free amplicon preparations derived from this construct were evaluated. Initial studies revealed that a single intramuscular (i.m.) injection of 10(6) infectious units (i.u.) of HSV:gp120 amplicon particles (HSV:gp120) elicited Env-specific cellular and humoral immune responses. A potent, CD8(+)-T-cell-mediated response to an H-2D(d)-restricted peptide from gp120 (RGPGRAFVTI) was measured by a gamma interferon ELISPOT and was confirmed by standard cytotoxic-T-lymphocyte assays. Immunoglobulin G enzyme-linked immunosorbent assay analysis showed the induction of a strong, Env-specific antibody response. An i.m. or an intradermal administration of HSV:gp120 at the tail base elicited a more potent cellular immune response than did an intraperitoneal (i.p.) inoculation, although an i.p. introduction generated a stronger humoral response. The immune response to HSV:gp120 was durable, with robust cellular and humoral responses persisting at 171 days after a single 10(6)-i.u. inoculation. The immune response to HSV:gp120 was also found to be dose dependent: as few as 10(4) i.u. elicited a strong T-cell response. Finally, HSV:gp120 elicited significant Env-specific cellular immune responses even in animals that had been previously infected with wild-type HSV-1. Taken together, these data strongly support the use of helper-free HSV-1 amplicon particles as vaccine delivery vectors.