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

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

Determination of HSV1-Tat and HSV1-LacZ viral attenuation in Balb/c mice.

<p>Groups of mice (n = 10) were infected by the intravaginal route with a single dose (ranging from 10⁴ to 10⁸ pfu/mouse) of HSV1-Tat or HSV1-LacZ at a single time point. Mice were observed daily for survival up to 20 days post-infection, for survival. The results of the group inoculated with 10⁸ pfu/mouse are shown. The results of one representative experiment (out of three) are shown. The Kaplan-Meier test was used to estimate the probability of clinical manifestation.</p

Proteolytic activity of proteasomes isolated from CB1 cells, uninfected or infected with 5 MOI of HSV1-Tat or HSV1-LacZ. Trypsin-like (A) and chymotrypsin-like (B) proteasome activities measured at 6, 12, and 24 h after infection are shown.

<p>Proteasome activity is expressed as arbitrary fluorescence units (AFU). The means (± SD) of three independent experiments are shown. The two-tailed Wilcoxon signed-rank test was used for statistical analysis, *p<0.05.</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

Analysis of the role of Tat protein on disease severity and survival of C57BL/6 mice.

<p>Mice (n = 5) were inoculated intravaginally with 10³ pfu/mouse of HSV1-LacZ and treated with (black dots) or without (grey dots) 5 µg/100 µl/mouse of Tat protein, administered intradermally at the same time. Control mice were inoculated with HSV1-LacZ alone. After 28 days mice were challenged intravaginally with a lethal dose of wild-type HSV1 and checked daily for the appearance of signs of disease (A) and survival (B). The two-tailed Mann-Whitney test was used for statistical analysis. **p<0.007.</p

Immunofluorescence micrographs at 24-infection of CB1 cells mock-infected (A and B, top panels) or infected with 1 (A) or 5 (B) MOI of HSV1-LacZ (middle panels) or of HSV1-Tat (bottom panels).

<p>Reactivity with anti-HSV1 antibodies is shown in red whereas DAPI-labelled nuclei are shown in blue in all panels.</p

Analysis of HSV1-specific T-cell responses in individual C57BL/6 mice.

<p>Mice were inoculated intravaginally with 10³ pfu/mouse of live attenuated HSV1-Tat or HSV1-LacZ. Seven days after infection, splenocytes were isolated from 5 mice/group and tested by ELISpot assay for IFN-γ (A) or IL-4 (B) cytokine production upon stimulation with SSI, ITA and QTF HSV-derived peptides. Results are expressed as number of spot-forming units (SFU)/million cells per mouse. Values at least 2-fold higher than the mean number of spots in the control wells (untreated cells), i.e., ≥50 SFU/million cells, were considered positive. The results of one representative experiment (out of three) are shown. The two-tailed Mann-Whitney test was used for statistical analysis, *p<0.05.</p

Vector representation and analysis of biological Tat expression.

<p>Schematic representation of the HSV1-LacZ/HSV1-Tat recombinant viruses (A). The black square in the HSV1 backbone indicates the UL41 locus (vhs gene), which has been deleted by insertion of the <i>lacZ-</i> or Tat-promoter cassette, respectively. The white squares represent the terminal and internal repeats of the HSV genome delimiting the unique regions (U_L: unique long; U_S: unique short). Analysis of Tat expression by HSV1-Tat recombinant vector (B). Western blot analysis of BALB/c cells infected with HSV1-Tat (1 MOI) after 6, 12 and 24 h post-infection. Negative controls are represented by uninfected cells and by cells infected with wild-type HSV1 or HSV1-LacZ. Recombinant Tat protein (Tat) was used as positive control. Analysis of biological Tat activity by CAT ELISA assay (C). CAT expression induced by functional Tat protein produced by the HSV1-Tat viral vector was measured 6, 12 and 24 h post-infection in HeLa3T1 cell extracts infected with HSV1-Tat (0.1 or 1 MOI). Negative controls were uninfected cells and cells infected with wild-type HSV1-LacZ. The means (± SD) of six independent experiments are shown.</p