The folding competence of HIV-1 Tat mediated by interaction with TAR RNA

<p>The trans-activator Tat protein of HIV-1 belongs to the large family of intrinsically disordered proteins (IDPs), and is known to recruit various host proteins for the transactivation of viral RNA synthesis. Tat protein interacts with the transactivator response RNA (TAR RNA), exhibiting RNA chaperone activities for structural rearrangement of interacting RNAs. Here, considering that Tat-TAR RNA interaction is mutually cooperative, we examined the potential role of TAR RNA as Chaperna – RNA that provides chaperone function to proteins - for the folding of HIV-1 Tat. Using EGFP fusion as an indirect indicator for folding status, we monitored Tat-EGFP folding in HeLa cells via time-lapse fluorescence microscopy. The live cell imaging showed that the rate and the extent of folding of Tat-EGFP were stimulated by TAR RNA. The purified Tat-EGFP was denatured and the fluorescence was monitored <i>in vitro</i> under renaturation condition. The fluorescence was significantly increased by TAR RNA, and the mutations in TAR RNA that affected the interaction with Tat protein failed to promote Tat refolding. The results suggest that TAR RNA stabilizes Tat as unfolded, but prevents it from misfolding, and maintaining its folding competence for interaction with multiple host factors toward its transactivation. The Chaperna function of virally encoded RNA in establishing proteome link at the viral-host interface provides new insights to as yet largely unexplored RNA mediated protein folding in normal and dysregulated cellular metabolism.</p

Baculovirus Displaying Hemagglutinin Elicits Broad Cross-Protection against Influenza in Mice

<div><p>The widespread influenza virus infection further emphasizes the need for novel vaccine strategies that effectively reduce the impact of epidemic as well as pandemic influenza. Conventional influenza vaccines generally induce virus neutralizing antibody responses which are specific for a few antigenically related strains within the same subtype. However, antibodies directed against the conserved stalk domain of HA could neutralize multiple subtypes of influenza virus and thus provide broad-spectrum protection. In this study, we designed and constructed a recombinant baculovirus-based vaccine, rBac-HA virus, that expresses full-length HA of pandemic H1N1 influenza virus (A/California/04/09) on the viral envelope. We demonstrated that repeated intranasal immunizations with rBac-HA virus induced HA stalk-specific antibody responses and protective immunity against homologous as well as heterosubtypic virus challenge. The adoptive transfer experiment shows that the cross-protection is conferred by the immune sera which contain HA stalk-specific antibodies. These results warrant further development of rBac-HA virus as a broad-protective vaccine against influenza. The vaccine induced protection against infection with the same subtype as well as different subtype, promising a potential universal vaccine for broad protection against different subtypes to control influenza outbreaks including pandemic.</p></div

Construction and characterization of rBac-HA virus.

<p>(A) A schematic diagram of baculovirus vector construct containing HA gene. The pFastBac dual vector has engineered to encode pH1N1 HA gene from A/California/04/09 under the control of the polyhedrin (PH) promoter. The recombinant baculovirus, rBac-HA virus, was generated using the Bac-to-Bac baculovirus expression system. (B) The presence of multimeric HA protein in the purified viral particle was confirmed by cross-linking and western blotting as described in the Materials and Methods (HA proteins indicated by the arrowhead). Virus particles corresponding to 3×10⁶ PFU were cross-linked, heated in loading buffer at 65°C (cross-linked samples) or 100°C (non-cross-linked samples), and loaded for each lane. (C) The expression level of HA protein on the baculovirus envelope was analyzed using flow cytometry. The Sf9 cells were infected with rBac-HA virus or Bac-control at MOI of 10. 48 h post-infection, the cells were analyzed by HA-specific polyclonal antibody to determine the expression of rBac-HA on the envelope. Uninfected cells used as a negative control. <i>Bac-ctrl</i>, Bac-control-infected cells; <i>rBac-HA</i>, rBac-HA-infected cells; <i>Uninfected</i>, uninfected cells.</p

Sera, BAL fluids and splenocytes from rBac-HA virus-immune mice for adoptive transfer study.

<p>BALB/c mice (n = 5/group) were immunized twice on day 0 and 16 with 3×10⁷ PFU of rBac-HA virus via intranasal route. Sera, BAL fluids and splenocytes were collected from the immunized mice 3 weeks after second vaccination. (A) Whole HA or HA stalk protein-specific IgG in the pooled sera and (B) HA-IgA or HA stalk-IgG in the pooled BAL fluids were measured by ELISA. The results indicate Log₂ end-point titers. (C) HA-specific CD8⁺ T cells (H-2K^d/HA_533-541 tetramer⁺, CD8⁺ and CD44⁺) were obtained from peripheral bood or lung tissue of boosted mice, and measured by flow cytometry. (D) Intracellular IFN-γ-producing CD8⁺ T cells (IFN-γ⁺, CD8⁺ and CD44⁺) from the donor lung tissue were measured by flow cytometry. “N.S.” indicates that there is no statistical significance between “PBS” group and “Bac-ctrl" group. *, Statistical significance to “Bac-control” (p<0.05). <i>N</i>.<i>S</i>., not significant; <i>Bac-ctrl</i>, Bac-control.</p

rBac-HA virus elicited stalk-specific Abs.

<p>BALB/c mice (n = 5/group) were immunized twice on day 0 and 16 with 3×10⁶ PFU or 3×10⁷ PFU of rBac-HA virus via intranasal route. Sera were collected from all mice 3 weeks after second vaccination. HA stalk-specific Abs were measured in boosted mice by ELISA using chimeric H9/1 protein [H9 HA head on top of an H1 (PR8) stalk domain]. The result was expressed absorbance at 450 nm. <i>O</i>.<i>D</i>., optical density; <i>Positive</i>, Sera from mice vaccinated with 3×10⁷ PFU of recombinant adenovirus encoding HA of H5N1 virus and ectodomain of matrix 2 protein (M2e) of H1N1 virus; <i>Bac-ctrl</i>, Bac-control.</p

Humoral immune response induced by intranasal rBac-HA virus immunization.

<p>BALB/c mice (n = 4/group) were immunized twice on day 0 and 16 with 1×10⁶ PFU or 3×10⁶ PFU of rBac-HA virus via intranasal route. Sera were collected from primed and boosted mice 2 and 3 weeks after vaccination, respectively. Control mice were immunized i.n. with 3×10⁶ PFU of Bac-control. The group of mice injected with PBS as negative control. (A) HA-specific and (B) Baculovirus-specific IgG antibody titers were measured in primed and boosted mice sera by ELISA, respectively. (C) Hemagglutination inhibition (HAI) titers against A/California/04/09 were measured in the sera obtained from boosted mice. (D) Mucosal HA-specific IgA in BAL fluid were measured 3 weeks after vaccination by ELISA. The results indicate Log₂ end-point titers. “N.S.” indicates that there is no statistical significance between “PBS” group and “Bac-ctrl" group. *, Statistical significance with “Bac-ctrl” group (p<0.05). ^‡, Statistical significance with “Priming” group (p<0.05). ^†, Statistical significance with “PBS” group (p<0.05). <i>N</i>.<i>S</i>., not significant; <i>Bac-ctrl</i>, Bac-control.</p

Adoptive transfer of sera from rBac-HA-immune mice provides protection against challenge with heterosubtypic H5N1 virus.

<p>BALB/c mice (n = 5/group) were immunized twice on day 0 and 16 with 3×10⁷ PFU of rBac-HA virus via intranasal route. Sera, BAL fluids and splenocytes collected from the immunized mice 3 weeks after second vaccination were adoptively transferred to naïve mice, and 24 h later these mice were challenged with 10 LD₅₀ of H5N1 influenza virus. Survival of recipient mice were monitored for 14 days after challenge. <i>Bac-ctrl</i>, Bac-control.</p

Mice immunized with rBac-HA virus show protection against challenge with heterosubtypic H5N1 virus.

<p>BALB/c mice (n = 5/group) were immunized twice on day 0 and 16 with 3×10⁶ PFU or 3×10⁷ PFU of rBac-HA virus via intranasal route. Each group of mice was challenged by intranasal administration with 50 LD₅₀ of H5N1 influenza virus. (A) Weight loss was monitored daily for 14 days after challenge. The results were expressed in percent body weight compared to beginning of the trial. (B) Survival of all group of mice were also monitored for 14 days. *, Statistical significance with “Bac-ctrl” group (p<0.05). <i>Bac-ctrl</i>, Bac-control.</p

Inactivated Eyedrop Influenza Vaccine Adjuvanted with Poly(I:C) Is Safe and Effective for Inducing Protective Systemic and Mucosal Immunity

<div><p>The eye route has been evaluated as an efficient vaccine delivery routes. However, in order to induce sufficient antibody production with inactivated vaccine, testing of the safety and efficacy of the use of inactivated antigen plus adjuvant is needed. Here, we assessed various types of adjuvants in eyedrop as an anti-influenza serum and mucosal Ab production-enhancer in BALB/c mice. Among the adjuvants, poly (I:C) showed as much enhancement in antigen-specific serum IgG and mucosal IgA antibody production as cholera toxin (CT) after vaccinations with trivalent hemagglutinin-subunits or split H1N1 vaccine antigen in mice. Vaccination with split H1N1 eyedrop vaccine antigen plus poly(I:C) showed a similar or slightly lower efficacy in inducing antibody production than intranasal vaccination; the eyedrop vaccine-induced immunity was enough to protect mice from lethal homologous influenza A/California/04/09 (H1N1) virus challenge. Additionally, ocular inoculation with poly(I:C) plus vaccine antigen generated no signs of inflammation within 24 hours: no increases in the mRNA expression levels of inflammatory cytokines nor in the infiltration of mononuclear cells to administration sites. In contrast, CT administration induced increased expression of IL-6 cytokine mRNA and mononuclear cell infiltration in the conjunctiva within 24 hours of vaccination. Moreover, inoculated visualizing materials by eyedrop did not contaminate the surface of the olfactory bulb in mice; meanwhile, intranasally administered materials defiled the surface of the brain. On the basis of these findings, we propose that the use of eyedrop inactivated influenza vaccine plus poly(I:C) is a safe and effective mucosal vaccine strategy for inducing protective anti-influenza immunity.</p></div

Comparison of antibody production by the amount of hemagglutinin (HA) or poly(I:C).

<p>(A). Dose-dependent HA vaccine antigens were administered with 10 μg poly(I:C) resolved in 5 μl of PBS by drops on both eyes two times at a 2-week interval in female BALB/c mice. HA-specific Ab levels were measured in serum and in various mucosal fluids 2 weeks after final vaccination by ELISA. (B) 1 μg of HA vaccine antigen plus dose-dependent poly(I:C) was vaccinated by drops on both eyes two times at a 2-week interval in female BALB/c mice. HA-specific Ab levels were measured in serum and in various mucosal secretions 2 weeks after final vaccination by ELISA. * <i>p</i> < 0.05; ** <i>p</i> < 0.01 versus Ag alone group; ‘n.s.’, non-significant. Results are representative of three independent experiments, with three mice in each group.</p