Proteins are poor immunogens that require an adjuvant to raise an immune response. Here we show that the human immunodeficiency virus, type 1 Tat protein possesses an autoadjuvant property, and we have identified the determinants and the molecular events that are associated with this unusual property. Using a series of chemically synthesized Tat101 derivatives, we show that the core region controls the autoadjuvant phenomenon independently of the B-cell recognition and T-cell stimulation that are associated with epitopes respectively located on the N-terminal region and the cysteine-rich region. We also show that cysteine-mediated oligomerization is a key molecular event of the adjuvant-free antibody response. In particular, a Tat dimer formed by the oxidation of two cysteine residues, at position 34 only, raises an adjuvant-free antibody response that is comparable with that observed with the wildtype protein. Unlike the parent protein, the Tat dimer has no transactivating activity and remains homogeneous for several weeks in solution. This construct might be of value for the design of an adjuvant-free Tat-based vaccine. Furthermore, we suggest that the specific autoadjuvanticity determinant of Tat could be used to provide other proteins with adjuvant-free immunogenicity.Most free proteins injected in a soluble form in humans or animals are poor immunogens and can induce B-or T-cell tolerance or rapid proliferation followed by rapid death of Ag-specific T-cells. They usually become immunogenic when they are mixed with an adjuvant. It is not completely understood how adjuvants are able to convert a tolerogenic stimulus into an immunogenic one. The process seems to be related to an increase in immunogen half-life through the so-called "depot effect" (1) and to a series of immunological events that include induction of inflammation and of inflammatory cytokines (2, 3), improvement of Ag delivery to Ag-presenting cells (4), increase in Ag processing and presentation through the induction of major histocompatibility complex and/or costimulatory molecules (5), and induction of the production of immunomodulatory cytokines (6). Because most proteins cannot trigger such complex events, it is no surprise that, alone, they do not induce an immune response.The Tat (transcriptional transactivator) protein of HIV-1 2 is a regulatory protein that is produced early after infection and that is essential for viral replication (7,8). This molecule is released in the extracellular milieu (9, 10), where it exhibits numerous biological activities. In particular, Tat was suggested to induce angiogenesis (11-13), chemotaxis of monocytes (14), and secretion of proinflammatory cytokines such as IL-1, IL-6, and tumor necrosis factor ␣ (15). Furthermore, Tat was proposed to target monocyte-derived dendritic cells and enhance their maturation, function, and Ag-specific T-cell responses (16). Also, a recent report showed that Tat can reprogram immature dendritic cells to express chemoattractants for activated T-cells and macrophages, but in...

Adenylate cyclase-hemolysin toxin (CyaA) produced from the human respiratory tract pathogen Bordetella pertussis requires fatty-acyl modification by CyaC-acyltransferase to become an active toxin. Previously, the recombinant CyaA pore-forming (CyaA-PF) fragment expressed in Escherichia coli was shown to be hemolytically active upon palmitoylation in vivo by cosynthesized CyaC. Here, the 21-kDa CyaC enzyme separately expressed in E. coli as an inclusion body was solubilized in 8 M urea and successfully refolded into an enzymatically active monomer. In addition to the capability of activating CyaA-PF in vitro, CyaC showed esterase activity against p-nitrophenyl acetate (pNPA) and p-nitrophenyl palmitate (pNPP), with preferential hydrolysis toward pNPP when compared with chymotrypsin. A homology-based CyaC structure suggested a conceivable role of a catalytic triad including Ser(30), His(33) and Tyr(66) in substrate catalysis. Alanine substitutions of these individual residues caused a drastic decrease in specific activities of all three mutant enzymes (S30A, H33A and Y66A) toward pNPP, signifying that CyaC-acyltransferase shares a similar mechanism of hydrolysis with a serine esterase in which Ser(30) is part of the catalytic triad.

ABSTRACT. Pasteurella multocida is a Gram-negative bacterium known to infect a wide range of domestic and wild animals. The increasing incidence of P. multocida isolated from cases of fowl cholera and hemorrhagic septicemia has led to a renewed interest in this pathogen as well as in the development of vaccines. In this study, PCR primers were designed to amplify the fragment of plpB gene from P. multocida FC-Pakchong (A:1). The purified PCR product of plpB gene consisting of 831 base pairs was inserted into the pGEX-5X-1 plasmid, which expressed the GST protein, and then transformed into E. coli. The purified fusion GST-PlpB protein showed a major band of about 63 kDa on SDS-PAGE gel. After enzyme cleavage, the recombinant PlpB protein appeared at the estimated size of 36 kDa. The recombinant GST-PlpB protein was tested for the toxicity in vivo. The results showed no toxicity in mice at the highest tested concentration of the protein. Moreover, the immunoprotective property of the recombinant GST-PlpB protein was determined in mice after subcutaneous immunization and challenge with an approximate dose of 50-100 LD 50 of P. multocida serotype A:1 and A:3,4. It was demonstrated that this subunit vaccine provided 20-30% survival rate after subcutaneous immunization and challenge with an approximate dose of 50-100 LD 50 of P. multocida serotype A:1 and A:3,4 whereas all of the non-immunized mice died from P. multocida infection. In conclusion, our data indicated that the PlpB protein may not be an appropriate target as a candidate subunit vaccine for P. multocida infection.KEY WORDS: 39-kDa outer membrane associated protein, fowl cholera, Pasteurella multocida, PlpB, subunit vaccine.