Fiorelli, M. Mice were immunized at days 0, 14, and 28, and given boosters after 15 weeks; sera were drawn 7 days after each booster, and the antibody titer was determined by enzyme-linked immunosorbent assay. All three immunization methods induced the development of a strong anti-Tat immunological response, which increased over time. Isotype subclass determination showed the presence of mucosal (immunoglobulin A) immunity soon after the beginning of the oral immunization protocol, and the data were confirmed by the presence of anti-Tat antibodies in fecal pellets and in vaginal washes. We also exhibited that sera from immunized mice inhibited with high efficiency recombinant Tat-dependent transactivation of the HIV-1 long terminal repeat promoter. This neutralization activity might be relevant for the suppression of extracellular Tat activities, which play an important role in HIV disease development. Human immunodeficiency computer virus (HIV) infection is responsible for a large number of deaths annually and represents a significant threat to global health. The development of an HIV vaccine is an urgent priority and represents the only realistic approach to control the global growth of the HIV pandemic, particularly Felbamate in the developing world. Although preventive immunity remains the main goal, secondary endpoints (e.g., block of computer virus replication and delay of disease onset) are being considered as more achievable aims. Considering that natural transmission of HIV occurs at the mucosae and that mucosa-associated lymphoid tissues may be the earliest target for computer virus replication (14, 36), successful induction of strong mucosal immunity may require vaccination by a mucosal route. Thus, a successful HIV vaccine may ultimately be a nonreplicating mucosal vaccine consisting of structural or nonstructural immunogens, alone or in combination. Over the last 2 decades, most efforts in HIV vaccine development have been based on the Felbamate use of the HIV envelope protein (Env), with the goal of inducing sterilizing immunity. However, Env-based vaccines have failed because of the complex structure of Env and the difficulty of generating broadly reactive, high-titer neutralizing antibodies as well as its high variability among viral isolates (20). In contrast, the Tat protein of HIV-1 shows little variability among HIV subtypes and is highly conserved in both inter- and intrapatient variants (11). Tat is usually a small regulatory protein composed of 86 to 101 amino acid residues (depending on the viral isolate) encoded by two exons. Several studies have dissected the molecular mechanisms of Tat function and shown that the protein acts as a powerful transcriptional activator of viral gene expression. At the long terminal repeat (LTR) promoter, the protein binds a was performed for 24 h at 19C in the dark with the addition of 100 mg/liter PP2Bgamma acetosyringone. Shoot regeneration was carried out in the presence of 300 mg/liter ticarcillin-clavulanic Felbamate acid (Timentin) to inhibit and 100 mg/liter kanamycin as a selective agent. The Patho Screen NptII enzyme-linked immunosorbent assay (ELISA) kit (AGDIA) was employed to detect expression following the Felbamate manufacturer’s instructions. Construction of plant expression vectors. Plasmid pBSHIVVAC was constructed by ligating the following components: (i) a 2.2-kb fragment containing the tomato E8 fruit-specific promoter obtained by EcoRI/BamHI digestion of pUC118 E8 (21); (ii) a 450-bp fragment encoding the Tat cDNA (provided by Mauro Giacca, ICGEB, Trieste, Italy), obtained by BamHI/XbaI digestion of pCMVTat101 (26); (iii) a XbaI/PstI 410-bp fragment made up of the cauliflower mosaic computer virus terminator; and (iv) pBlueScript II SK(+) Felbamate (Stratagene) digested with PstI/EcoRI. The correct orientation of all components was verified by restriction mapping and sequencing using an ABI PRISM Big Dye terminator system (Applied Biosystems). Subsequently, the whole cassette was excised from pBlueScript and subcloned, as a KpnI/SstI fragment, into the binary vector pCambia 2301 (www.cambia.org), which confers resistance to kanamycin. This vector was called pHIVVAC-K (Fig. ?(Fig.1A).1A). Plasmids were managed and multiplied in XL1-Blue. The pHIVVAC-K vector was transferred into.