Recombinant HIV-1 vaccine candidates based on replication-defective flavivirus vector.

Multiple approaches utilizing viral and DNA vectors have shown promise in the development of an effective vaccine against HIV. In this study, an alternative replication-defective flavivirus vector, RepliVax (RV), was evaluated for the delivery of HIV-1 immunogens. Recombinant RV-HIV viruses were engineered to stably express clade C virus Gag and Env (gp120TM) proteins and propagated in Vero helper cells. RV-based vectors enabled efficient expression and correct maturation of Gag and gp120TM proteins, were apathogenic in a sensitive suckling mouse neurovirulence test, and were similar in immunogenicity to recombinant poxvirus NYVAC-HIV vectors in homologous or heterologous prime-boost combinations in mice. In a pilot NHP study, immunogenicity of RV-HIV viruses used as a prime or boost for DNA or NYVAC candidates was compared to a DNA prime/NYVAC boost benchmark scheme when administered together with adjuvanted gp120 protein. Similar neutralizing antibody titers, binding IgG titers measured against a broad panel of Env and Gag antigens, and ADCC responses were observed in the groups throughout the course of the study, and T cell responses were elicited. The entire data demonstrate that RV vectors have the potential as novel HIV-1 vaccine components for use in combination with other promising candidates to develop new effective vaccination strategies.

Identification of rat prostatic steroid-binding protein as a target antigen of experimental autoimmune prostatitis: implications for prostate cancer therapy.

The long term goal of this study is to develop autoimmune prostatitis as a therapy for prostate cancer. An immune attack capable of destroying normal prostate epithelial cells should also destroy malignant prostate tissue and provide therapeutic benefit in cancer patients. The current study was initiated to identify antigenic targets for experimental autoimmune prostatitis on the assumption that such proteins might also be suitable targets for immunotherapy of prostate cancer. Male Lewis rats were immunized with syngeneic prostate homogenates, and the immune sera were used to screen prostate proteins for immunoreactivity by Western blot analysis. The dominant protein recognized by the immune sera was purified by ion exchange chromatography and reverse phase HPLC. Microsequence analysis of two polypeptide components of this immunodominant protein demonstrated N-terminal sequences identical with two of the three component chains of rat prostatic steroid-binding protein (PSBP). T cell responses to PSBP were also detected in rats immunized with prostate homogenate. Immunizing male rats with purified PSBP induced vigorous Ab and T cell responses. Significant prostate inflammation was observed in some rats immunized with PSBP. Adoptive transfer of T cells immune to PSBP induced rapid and severe destructive autoimmune prostatitis. These results demonstrate that PSBP is a major target Ag of experimental autoimmune prostatitis in a rat model and may serve as a target Ag for vaccine and T cell therapy against prostate cancer.

Differential regulation of proto-oncogenes c-jun and c-fos in T lymphocytes activated through CD28.

The T cell surface molecule CD28 binds to ligands on accessory cells and APCs, playing an important costimulatory role in the response of T cells to Ags. Our knowledge of the intracellular signaling pathways coupled to this receptor is incomplete. In addition to activation of phospholipase C gamma 1, ligation of this receptor also seems to activate a calcium-independent, CD28-specific pathway. In this paper, we report that cross-linking of CD28 (but not CD2, CD5, LFA-1, or CD7) leads to an elevation of c-jun mRNA, with only minimal activation of c-fos expression. CD28-dependent induction of c-jun expression requires protein tyrosine kinase activity, but does not depend on activation of a phorbol ester-responsive protein kinase C or elevation of cytosolic calcium. Furthermore, CD28-dependent elevation of c-jun mRNA does not appear to be mediated at the level of mRNA stability. A mechanism is suggested whereby expression of c-jun and junB, in the absence of members of the fos family, can prevent inappropriate activation of T cells caused by ligation of CD28 in the absence of a specific antigenic stimulus.

Bovine interleukin 2: regulatory mechanisms.

A cDNA clone of the bovine interleukin-2 (IL-2) gene has been isolated and demonstrated to produce a functional bovine IL-2 protein when transfected into either CV-1 or COS-1 monkey cells. Homology comparisons of both the nucleotide and predicted amino acid sequences of bovine IL-2 with those of the human and mouse show extensive regions of sequence conservation between the species. The amino acid sequence of the mature bovine IL-2 protein shares about 60-63% homology with those of the human and mouse, but the 3' untranslated regions of the human and mouse gene share as much, if not greater, sequence homology with the 3' untranslated regions of the human and mouse genes. In particular, a tandemly repeated sequence (TATT), n, found in the 3' untranslated tail of the bovine IL-2 clone is also found in the 3' untranslated region of a large group of cytokine genes and other inducible genes of the lymphoid and immune response systems. This sequence may serve a specific regulatory function in the immune system.

Molecular cloning of a functional bovine interleukin 2 cDNA.

A cDNA clone of the bovine interleukin 2 (IL-2) gene has been isolated and demonstrated to be functional in the production of secreted bovine IL-2 protein when transfected into monkey cells. The bovine IL-2 clone is 791 base pairs in length and contains an open reading frame of 474 base pairs coding for a bovine IL-2 precursor polypeptide of 158 amino acids with an estimated molecular weight of 17,884. The putative hydrophobic leader or signal sequence of the precursor protein is 23 amino acid residues long, suggesting that, after removal by processing, the mature secreted bovine IL-2 protein contains 135 amino acids and has a molecular weight of 15,464. Comparisons of both the nucleotide sequence and the predicted amino acid sequence of bovine IL-2 with those of the human and mouse IL-2 show extensive regions of sequence conservation between the species, interspersed with other regions of less similarity. The 3' untranslated region of the bovine IL-2 gene shares as much, if not greater, sequence homology with the 3' untranslated regions of the human and mouse genes as do the transcribed coding regions of these genes, suggesting an involvement of this region in regulation. In particular, a tandemly repeated sequence, (TATT)n, found in the 3' untranslated tail of the bovine IL-2 clone is also found in the 3' untranslated region of the other known interleukin and interferon genes, as well as in similar regions of many other inducible genes of the lymphoid and immune response systems, suggesting a cell or tissue-specific regulatory function for these evolutionarily conserved sequences.

Antibacterial hemolymph proteins of Manduca sexta.

Exclusion column fractionated immune hemolymph of the M. sexta larva contains five peaks of anti-E. coli activity with molecular weights of greater than 140 kD and approximately 91, 54, 14 and 4 kD, plus one peak of lysozyme activity with a molecular weight of 17 kD. Purification of the 54 kD peak showed that this peak consists of the previously described M18 proteins which have monomeric weights of approximately 20 kD and had antibacterial activity against certain gram negative bacteria. Approximately 80% of the total hemolymph antibacterial activity was detected in the 14 and 4 kD peaks. These proteins, which kill both gram negative and gram positive bacteria, appeared to be directly analogous to the cecropins of H. cecropia. The greater than 140 and 91 kD peaks constituted only a minor part of the total antibacterial activity.

Effect of sublethal Bacillus thuringiensis crystal endotoxin treatment on the larval midgut of a moth, Manduca: SEM study.

The effect of a single, sublethal dose of B. thuringiensis crystal endotoxin on the midgut of the moth Manduca sexta larvae was monitored during acute and recovery stages. Initially both goblet and columnar cells swelled. Many columnar cells produced membrane extrusions. In some cases the affected cells ruptured, extruding cellular debris into the midgut lumen. Following the acute stage, the midgut tissue recovered, the damaged cells being extruded into the midgut lumen apparently as newly regenerated cells rose to take their place. The insects appeared to recover completely and continue normal development.