Tat-mediated CD8⁺ T cell activation.

<p>(<b>A</b>) Fresh splenocytes from C57BL/6 mice were co-cultured, in the presence or absence of 1µg/ml of Tat, with autologous splenocytes previously loaded with the SSI peptide epitope. After 8 days, cells were assayed in IFNγ Elispot. One representative experiment out of five is shown. (<b>B</b>) Splenocytes were purified from HSV1-infected C57BL/6 mice at day 8 post-infection and assayed in IFNγ Elispot against the SSI epitope in the presence or absence of 1 µg/ml of Tat. One representative experiment out of five is shown. (<b>C</b>) Balb/c mice (3 per groups) were injected with 5 µg of Gag alone or in combination with 5 µg of Tat. Ten days after vaccination mice were sacrificed and fresh splenocytes assayed in IFNγ Elispot against the indicated Gag-derived T cell peptide epitopes (see also <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0077746#pone.0077746.s002" target="_blank">Table S1</a>). One representative experiment out of three is shown.</p

Tat administered at the time of antigen-priming favors an effector memory phenotype.

<p>Control and Tat-treated mice were infected with HSV1 wt (<b>A</b> and <b>B</b>) or with replicative-defective HSV1 (<b>C</b> and <b>D</b>) and sacrificed at days 70 post-infection. (<b>A</b> and <b>C</b>) Percentage of SSI-specific CD8⁺ T cells detected by dextramer staining. (<b>B</b> and <b>D</b>) CD62L expression was measured by flow cytometry on SSI-specific CD8⁺ T cells. Data are presented as mean ± SEM of 5 mice per group. For statistical analysis two-tailed Mann Whitney test was used. *P<0.05. One representative experiment out of three is shown.</p

Tat does not contribute to the control of HSV1 acute infection.

<p>Control and Tat-treated HSV1-infected C57/BL6 mice were checked daily for the appearance of disease signs. (<b>A</b>) Mean of disease scores ± SEM of 20 mice per group is shown. For statistical analysis two-tailed Mann Whitney test was used. **P<0.01. (<b>B</b>) Probability of developing disease signs is shown for each group. Figure represents Kaplan-Meier estimation of the probability of clinical manifestations. For statistical analysis Log rank test was used. One representative experiment out of three is shown.</p

Tat administered at the time of antigen-priming favors a Th1 profile of the humoral response.

<p>Blood samples from control and Tat-treated HSV1-infected mice were collected and the presence of anti-HSV1 antibodies was detected by ELISA assay. (<b>A</b>) Anti-HSV1 IgG1 and IgG2a were measured at days 20 (left) and 70 (right) post-infection. (<b>B</b>) Total anti-HSV1 IgG were measured at days 20 (left) and 70 (right) post-infection. Data are presented as mean ± SEM of 5 mice per group. For statistical analysis two-tailed Mann Whitney test was used. *P<0.05. One representative experiment out of three is shown.</p

Effects of RAMB treatment on the levels of polyubiquitinated proteins in HeLa cells.

<p><i>Left panel:</i> immunoblot analysis of ubiquitinated proteins in HeLa cells after 6 hours exposure with or without 10 µM RAMBs. Bortezomib was used as positive control. Equal protein loading in each lane was verified by using an antibody against GAPDH. <i>Right panel:</i> Quantification of the Ubiquitin/GAPDH ratios.</p

An Attenuated Herpes Simplex Virus Type 1 (HSV1) Encoding the HIV-1 Tat Protein Protects Mice from a Deadly Mucosal HSV1 Challenge

<div><p>Herpes simplex virus types 1 and 2 (HSV1 and HSV2) are common infectious agents in both industrialized and developing countries. They cause recurrent asymptomatic and/or symptomatic infections, and life-threatening diseases and death in newborns and immunocompromised patients. Current treatment for HSV relies on antiviral medications, which can halt the symptomatic diseases but cannot prevent the shedding that occurs in asymptomatic patients or, consequently, the spread of the viruses. Therefore, prevention rather than treatment of HSV infections has long been an area of intense research, but thus far effective anti-HSV vaccines still remain elusive. One of the key hurdles to overcome in anti-HSV vaccine development is the identification and effective use of strategies that promote the emergence of Th1-type immune responses against a wide range of epitopes involved in the control of viral replication. Since the HIV1 Tat protein has several immunomodulatory activities and increases CTL recognition of dominant and subdominant epitopes of heterologous antigens, we generated and assayed a recombinant attenuated replication-competent HSV1 vector containing the <i>tat</i> gene (HSV1-Tat). In this proof-of-concept study we show that immunization with this vector conferred protection in 100% of mice challenged intravaginally with a lethal dose of wild-type HSV1. We demonstrate that the presence of Tat within the recombinant virus increased and broadened Th1-like and CTL responses against HSV-derived T-cell epitopes and elicited in most immunized mice detectable IgG responses. In sharp contrast, a similarly attenuated HSV1 recombinant vector without Tat (HSV1-LacZ), induced low and different T cell responses, no measurable antibody responses and did not protect mice against the wild-type HSV1 challenge. These findings strongly suggest that recombinant HSV1 vectors expressing Tat merit further investigation for their potential to prevent and/or contain HSV1 infection and dissemination.</p></div

RAMB1 treatment prevents anchorage-dependent colony formation and synergizes with Chloroquine to kill cervical cancer cells.

<p>A. Equal numbers of SiHa and Caski cells (10³) were seeded into 6-wells plastic dishes and treated with or without RAMB1 at the indicated concentrations over a period of 10 days. Colonies were visualized by crystal violet staining. B. <i>Left panel</i>: Quantification of colony number in mock versus RAMB1 treated HeLa cells. <i>Middle panel</i>: Quantification of colony size in mock versus RAMB1 treated cervical cancer cells. <i>Right panel</i>: Representative experiment of HeLa cells (10²) which were seeded into 6-wells plastic dishes and treated with or without RAMB1 at the indicated concentration over a period of 10 days. Colonies were visualized by crystal violet staining and manually counted using an inverted microscope. <i>Inserts:</i> Representative example of colony size in mock versus RAMB1-treated cells. <i>Middle panel:</i> Quantification of colony number. <i>Right panel</i>: Quantification of colony size, representative of three independent experiments. **, P<0.02. C. CaSki cells were treated with checkerboard dilution series of RAMB1 and Chloroquine over a period of 24 hours. Cell viability was measured by XTT assay and calculated as percent of control untreated cultures. Synergy is expressed as combination index (CI).</p

Evaluation of anti-HSV1 specific antibody titers (IgG, IgG₁, and IgG_2a) in sera from C57BL/6 (A) or BALB/c (B) mice treated by the intravaginal route with 10³ pfu/mouse of HSV1-Tat or HSV1-LacZ.

<p>Twenty days after infection, sera from 5 mice/group were collected by eye-bleeding, and anti-HSV1 antibody responses were measured by ELISA. The results of one representative experiment (out of three) are shown. The Fisher exact test was used for statistical analysis. *P<0.05.</p