RIG-I and MDA-5 detection of viral RNA-dependent RNA polymerase activity restricts positive-strand RNA virus replication.

Type I interferons (IFN) are important for antiviral responses. Melanoma differentiation-associated gene 5 (MDA-5) and retinoic acid-induced gene I (RIG-I) proteins detect cytosolic double-stranded RNA (dsRNA) or 5'-triphosphate (5'-ppp) RNA and mediate IFN production. Cytosolic 5'-ppp RNA and dsRNA are generated during viral RNA replication and transcription by viral RNA replicases [RNA-dependent RNA polymerases (RdRp)]. Here, we show that the Semliki Forest virus (SFV) RNA replicase can induce IFN-ß independently of viral RNA replication and transcription. The SFV replicase converts host cell RNA into 5'-ppp dsRNA and induces IFN-ß through the RIG-I and MDA-5 pathways. Inactivation of the SFV replicase RdRp activity prevents IFN-ß induction. These IFN-inducing modified host cell RNAs are abundantly produced during both wild-type SFV and its non-pathogenic mutant infection. Furthermore, in contrast to the wild-type SFV replicase a non-pathogenic mutant replicase triggers increased IFN-ß production, which leads to a shutdown of virus replication. These results suggest that host cells can restrict RNA virus replication by detecting the products of unspecific viral replicase RdRp activity.

Induction of human immunodeficiency virus type-1-specific immunity with a novel gene transport unit (GTU)-MultiHIV DNA vaccine.

A multiHIV fusion gene expressing an antigenic fusion protein composed of regulatory HIV-1 proteins Rev, Nef, and Tat, as well as Gag p17/p24 and a stretch of 11 cytotoxic T lymphocyte (CTL) epitope clusters from Pol and Env, was cloned into a novel DNA vector named the Gene Transport Unit (GTU). A mouse H-2(d)-restricted HIV-1 gp120 epitope (RGPGRAFVTI) was cloned into the fusion gene as well. In addition to the HIV- 1 genes the GTU codes for a nuclear anchoring protein (bovine papilloma virus E2), ensuring the long maintenance of the vector and a high expression level of the selected immunogens. BALB/c mice were immunized with the GTU-MultiHIV DNA construct by different routes and regimens of immunization to assess the immunogenicity of the DNA vaccine in vivo. Mice developed strong CD8(+) CTL responses to HIV-1 Env and Gag measured by an ELISPOT-IFN-gamma assay and chromium release assay. In addition, T cell responses to regulatory proteins Rev, Nef, and Tat were induced. Antibody responses were detected to each of the HIV antigens encoded by the DNA construct. Minimal doses of the GTU-MultiHIV DNA delivered by gene gun were potent in inducing significant HIV-specific CTL responses. The equivalent doses of the conventional plasmid expressing MultiHIV DNA delivered by gene gun failed to do so. An ideal DNA vaccine should yield high expression of the viral antigens for a prolonged period of time, and expression of the multiple viral antigens is probably required for the induction of a broad and protective immune response. The GTU-MultiHIV DNA vaccine described is a good vaccine candidate that meets the above criteria.

Therapeutic immunisation plus cytokine and hormone therapy improves CD4 T-cell counts, restores anti-HIV-1 responses and reduces immune activation in treated chronic HIV-1 infection.

BACKGROUND: This randomised, open label, phase I, immunotherapeutic study investigated the effects of interleukin (IL)-2, granulocyte-macrophage colony-stimulating factor (GM-CSF), recombinant human growth hormone (rhGH), and therapeutic immunisation (a Clade B DNA vaccine) on combination antiretroviral therapy (cART)-treated HIV-1-infected individuals, with the objective to reverse residual T-cell dysfunction. METHODS: Twelve HIV-1(+) patients on suppressive cART with baseline CD4 T-cell counts >400 cells/mm(3) blood were randomised into one of three groups: (1) vaccine, IL-2, GM-CSF and rhGH (n=3); (2) vaccine alone (n=4); or (3) IL-2, GM-CSF and rhGH (n=5). Samples were collected at weeks 0, 1, 2, 4, 6, 8, 12, 16, 24 and 48. Interferon (IFN)-γ, IL-2, IL-4 and perforin ELISpot assays performed at each time point quantified functional responses to Gag p17/p24, Nef, Rev, and Tat peptides; and detailed T-cell immunophenotyping was undertaken by flow cytometry. Proviral DNA was also measured. RESULTS: Median baseline CD4 T-cell count was 757 cells/mm(3) (interquartile range [IQR] 567-886 cells/mm(3)), median age 48 years (IQR 42-51 years), and plasma HIV-1-RNA <50 copies/ml for all subjects. Patients who received vaccine plus IL-2, GM-CSF and rhGH (group 1) showed the most marked changes. Assessing mean changes from baseline to week 48 revealed significantly elevated numbers of CD4 T cells (p=0.0083) and improved CD4/CD8 T-cell ratios (p=0.0033). This was accompanied by a significant reduction in expression of CD38 on CD4 T cells (p=0.0194), significantly increased IFN-γ and IL-2 production in response to Gag (p=0.0122) and elevated IFN-γ production in response to Tat (p=0.041) at week 48 compared to baseline. Subjects in all treatment groups showed significantly reduced PD-1 expression at week 48 compared to baseline, with some reductions in proviral DNA. CONCLUSIONS: Multifarious immunotherapeutic approaches in the context of fully suppressive cART further reduce immune activation, and improve both CD4 T-lymphocyte counts and HIV-1-specific T-cell responses (NCT01130376).

Elicitation of broad CTL response against HIV-1 by the DNA vaccine encoding artificial multi-component fusion protein MultiHIV--study in domestic pigs.

Broad CTL response against HIV-1 is one factor that helps to control the viral replication. We have constructed a DNA vaccine that encodes a large artificial fusion protein (MultiHIV) and shown it to be immunogenic in mice, swine and macaques. Inbred mice revealed CTL response only against two epitopes due to limited MHC class I variability. To assess the quality of the CTL response we addressed this question in domestic swine. Number of presented epitopes varied between 7 and 14 among the five selected animals. Epitopes detected in swine are localised in the same antigenic regions recognised in humans. This can be explained by the fact that swine MHC-I (SLA-I) complex is remarkably similar to human HLA-I. These results also indicate that immunogenicity profile of vaccines in domestic swine may predict the outcome of human immunisation.

Persistent immune responses induced by a human immunodeficiency virus DNA vaccine delivered in association with electroporation in the skin of nonhuman primates.

Strategies to improve vaccine efficacy are still required, especially in the case of chronic infections, including human immunodeficiency virus (HIV). DNA vaccines have potential advantages over conventional vaccines; however, low immunological efficacy has been demonstrated in many experiments involving large animals and in clinical trials. To improve the immunogenicity of DNA vaccines, we have designed a plasmid vector exploiting the binding capacity of the bovine papillomavirus E2 protein and we have used electroporation (EP) to increase DNA uptake after intradermal inoculation. We demonstrated, in nonhuman primates (NHPs), efficient induction of anti-HIV immunity with an improved DNA vaccine vector encoding an artificial fusion protein, consisting of several proteins and selected epitopes from HIV-1. We show that a DNA vaccine delivery method combining intradermal injection and noninvasive EP dramatically increased expression of the vaccine antigen selectively in the epidermis, and our observations strongly suggest the involvement of Langerhans cells in the strength and quality of the anti-HIV immune response. Although the humoral responses to the vaccine were transient, the cellular responses were exceptionally robust and persisted, at high levels, more than 2 years after the last vaccine boost. The immune responses were characterized by the induction of significant proportions of T cells producing both interferon-gamma and interleukin-2 cytokines, in both subpopulations, CD4(+) and CD8(+). This strategy is an attractive approach for vaccination in humans because of its high efficacy and the possible use of newly developed devices for EP.

GTU-MultiHIV DNA vaccine results in protection in a novel P815 tumor challenge model.

A novel animal model for testing the immunogenicity and protective immune response induced by HIV-1 DNA vaccines was developed. DBA/2 mice were immunized with GTU-MultiHIV DNA encoding multigene for Rev, Nef, Tat, optp17/24 and a stretch of Pol/Env epitopes. A single GTU-MultiHIV B-clade specific plasmid or Auxo-GTU-MultiHIV(mix) (mixture of four plasmids with A, B, C and FGH clade specific MultiHIV antigens) were administered via gene gun and cell-mediated and humoral immune responses were analysed. The protective efficacy of the immune response was evaluated by challenging the mice with syngeneic tumor cells (P815) stably transfected with the MultiHIV fusion gene. Our results show that the strong MultiHIV-specific immune response generated by the GTU-MultiHIV vaccines in DBA/2 mice was able to delay the tumor growth substantially, indicating that the CTL response detected in vitro confers protection in vivo. The model described here is a safe and feasible in vivo assay for assessment of the vaccine potency to induce protective cell-mediated immune responses.

CD43 has a functional NLS, interacts with beta-catenin, and affects gene expression.

CD43 is a transmembrane molecule with a highly O-glycosylated extracellular domain of mucin type. It is a normal constituent of leukocytes and found in colon adenoma, but not in normal colon epithelia. Here it is shown that the cytoplasmic tail of CD43 contains a functional bipartite nuclear localization signal directing it to the nucleus. The intracellular domain of CD43 interacts with beta-catenin and causes an upregulation of the beta-catenin target genes c-MYC and CyclinD1. The present results suggest that CD43 can be involved in nuclear signaling and via beta-catenin interaction be involved in cell proliferation.