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 modulates the kinetics and the magnitude of CTL responses.

<p>Splenocytes were purified from control and Tat-treated HSV1-infected C57/BL6 mice at days 4, 5, 6, 7, 8, 10 and 13 post-infection. (<b>A</b>) Percentage of SSI-specific CD8⁺ T cells detected by dextramer staining. Data are presented as mean ± SEM of 5 mice per group. One representative experiment out of three is shown. (<b>B</b>) Kinetics of SSI- and QTF-specific cellular responses detected by IFNγ Elispot on fresh splenocytes. Data are presented as mean ± SEM of 5 mice per group (left panel). Expansion and contraction were normalized, for each group, to values detected at day 7 (right panel). One representative experiment out of three is shown. (<b>C</b>) SSI- and QTF- specific IFNγ responses at days 5 and 6 post-infection. (<b>D</b>) SSI- and QTF- specific IFNγ responses at day 7 post-infection. (<b>E</b>) SSI- and QTF-specific IFNγ responses at day 13 post-infection. For statistical analysis two-tailed Mann Whitney test was used. *P<0.05. </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

Tat does not activate bystander T cells.

<p>Control and Tat-treated HSV1-infected C57/BL6 mice were sacrificed at days 4, 6, 8, 10 and 13 post-infection. CD8⁺ (<b>A</b>), CD4⁺ (<b>C</b>) and B (<b>D</b>) lymphocytes numbers were measured by flow cytometry. 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. (<b>B</b>) CD95 expression was measured by flow cytometry on CD8⁺ T cells. One representative experiment out of five is shown.</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

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

Evaluation of HSV1-Tat and HSV1-LacZ viral yields in vitro^a.

a<p>The replication of wild-type HSV1, HSV1-Tat and HSV1-LacZ viruses was evaluated <i>in vitro</i> using Vero cells (1×10⁶) infected, in monolayer or in suspension, with each virus at 0.1 or 1 MOI. Infected cells were harvested at 18 h post-infection, and viral production was assayed by means of the plaque assay method. Yields are expressed as the means of two independent experiments.</p

Disease severity and survival of BALB/c (A and B) or C57BL/6 (C and D) mice inoculated intravaginally with HSV1-LacZ or HSV1-Tat after challenge with a lethal dose of wild-type HSV1.

<p>Ten mice/group were inoculated intravaginally with 10³ pfu/mouse of HSV1-LacZ, HSV1-Tat or control buffer, and, 28 days later, challenged intravaginally with a lethal dose of wild-type HSV1 (2×10⁶ pfu/mouse for BALB/c mice and 2×10⁸ pfu/mouse for C57BL/6 mice). Mice were observed daily for appearance of signs of HSV1 disease and death. Disease severity scores (A for BALB/c and C for C57BL/6) and survival (B for BALB/c and D for C57BL/6) were assessed. Mean disease scores (± SD) for each group is shown. The results of one representative experiment (out of three) are shown. Data were analysed statistically using the two-tailed Mann Whitney test (panels A and C) and the Kaplan-Meier test. **P<0.01 (panels B and D).</p