TLR3 agonist and CD40-targeting vaccination induces immune responses and reduces HIV-1 reservoirs.

Activation of HIV-1 reservoirs and induction of anti-HIV-1 T cells are critical to control HIV-1 rebound after combined antiretroviral therapy (cART). Here we evaluated in humanized mice (hu-mice) with persistent HIV-1 infection the therapeutic effect of TLR3 agonist and a CD40-targeting HIV-1 vaccine, which consists of a string of 5 highly conserved CD4+ and CD8+ T cell epitope-rich regions of HIV-1 Gag, Nef, and Pol fused to the C-terminus of a recombinant anti-human CD40 antibody (αCD40.HIV5pep). We show that αCD40.HIV5pep vaccination coadministered with poly(I:C) adjuvant induced HIV-1-specific human CD8+ and CD4+ T cell responses in hu-mice. Interestingly, poly(I:C) treatment also reactivated HIV-1 reservoirs. When administrated in therapeutic settings in HIV-1-infected hu-mice under effective cART, αCD40.HIV5pep with poly(I:C) vaccination induced HIV-1-specific CD8+ T cells and reduced the level of cell-associated HIV-1 DNA (or HIV-1 reservoirs) in lymphoid tissues. Most strikingly, the vaccination significantly delayed HIV-1 rebound after cART cessation. In summary, the αCD40.HIV5pep with poly(I:C) vaccination approach both activates replication of HIV-1 reservoirs and enhances the anti-HIV-1 T cell response, leading to a reduced level of cell-associated HIV-1 DNA or reservoirs. Our proof-of-concept study has significant implication for the development of CD40-targeting HIV-1 vaccine to enhance anti-HIV-1 immunity and reduce HIV-1 reservoirs in patients with suppressive cART.

Broad and cross-clade CD4+ T-cell responses elicited by a DNA vaccine encoding highly conserved and promiscuous HIV-1 M-group consensus peptides.

T-cell based vaccine approaches have emerged to counteract HIV-1/AIDS. Broad, polyfunctional and cytotoxic CD4(+) T-cell responses have been associated with control of HIV-1 replication, which supports the inclusion of CD4(+) T-cell epitopes in vaccines. A successful HIV-1 vaccine should also be designed to overcome viral genetic diversity and be able to confer immunity in a high proportion of immunized individuals from a diverse HLA-bearing population. In this study, we rationally designed a multiepitopic DNA vaccine in order to elicit broad and cross-clade CD4(+) T-cell responses against highly conserved and promiscuous peptides from the HIV-1 M-group consensus sequence. We identified 27 conserved, multiple HLA-DR-binding peptides in the HIV-1 M-group consensus sequences of Gag, Pol, Nef, Vif, Vpr, Rev and Vpu using the TEPITOPE algorithm. The peptides bound in vitro to an average of 12 out of the 17 tested HLA-DR molecules and also to several molecules such as HLA-DP, -DQ and murine IA(b) and IA(d). Sixteen out of the 27 peptides were recognized by PBMC from patients infected with different HIV-1 variants and 72% of such patients recognized at least 1 peptide. Immunization with a DNA vaccine (HIVBr27) encoding the identified peptides elicited IFN-γ secretion against 11 out of the 27 peptides in BALB/c mice; CD4(+) and CD8(+) T-cell proliferation was observed against 8 and 6 peptides, respectively. HIVBr27 immunization elicited cross-clade T-cell responses against several HIV-1 peptide variants. Polyfunctional CD4(+) and CD8(+) T cells, able to simultaneously proliferate and produce IFN-γ and TNF-α, were also observed. This vaccine concept may cope with HIV-1 genetic diversity as well as provide increased population coverage, which are desirable features for an efficacious strategy against HIV-1/AIDS.

The HIV-1 Vpu viroporin inhibitor BIT225 does not affect Vpu-mediated tetherin antagonism.

Among its many roles, the HIV-1 accessory protein Vpu performs a viroporin function and also antagonizes the host cell restriction factor tetherin through its transmembrane domain. BIT225 is a small molecule inhibitor that specifically targets the Vpu viroporin function, which, in macrophages, resulted in late stage inhibition of virus release and decreased infectivity of released virus, a phenotype similar to tetherin-mediated restriction. Here, we investigated whether BIT225 might mediate its antiviral function, at least in part, via inhibition of Vpu-mediated tetherin antagonism. Using T-cell lines inducible for tetherin expression, we found that BIT225 does not exert its antiviral function by inhibiting Vpu-mediated tetherin downmodulation from the cell surface, the main site of action of tetherin activity. In addition, results from a bioluminescence resonance energy transfer (BRET) assay showed that the Vpu-tetherin interaction was not affected by BIT225. Our data provide support for the concept that tetherin antagonism and viroporin function are separable on the Vpu transmembrane and that viroporin function might be cell-type dependent. Further, this work contributes to the characterization of BIT225 as an inhibitor that specifically targets the viroporin function of Vpu.

Background K(2P) channels KCNK3/9/15 limit the budding of cell membrane-derived vesicles.

The main function of background two-pore potassium (K(2P)) channels KCNK3/9/15 is to stabilize the cell membrane potential. We previously observed that membrane potential depolarization enhances the release of HIV-1 viruses. Because membrane polarization affects the biomembrane directly, here we examined the effects of KCNK3/9/15 on the budding of nonviral vesicles. We found that depolarization by knocking down endogenous KCNK3/9/15 promoted secretion of cell-derived vesicles. We further used Vpu (an antagonist of KCNK3) as a model for the in vivo study of depolarization-stimulated secretion. Vpu is a HIV-1-encoded, ion channel-like protein (viroporin) capable of enhancing virus release and depolarizing the cell membrane potential. We found that Vpu could also promote nonviral vesicle release, perhaps through a similar mechanism that Vpu utilizes to promote viral particle release. Notably, T cells expressing Vpu alone became pathologically low in intracellular K(+) and insensitive to extracellular K(+) or membrane potential stimulation. In contrast, heterologous expression of KCNK3 in T cells stabilized the cell potentials by maintaining intracellular K(+). We thus concluded that KCNK3/9/15 expression limits membrane depolarization and depolarization-induced secretion at least in part by maintaining intracellular K(+).

Inhibition of ß-TrcP-dependent ubiquitination of p53 by HIV-1 Vpu promotes p53-mediated apoptosis in human T cells.

HIV-1 viral protein U (Vpu) is involved in ubiquitination and degradation of BM stromal cell Ag 2 and surface receptor CD4 through their recruitment to SCF(ß-TrcP) (Skp1/Cul1/F-box) ubiquitin ligase (SCF) complex. Here, we show that specific interaction of wild-type Vpu protein with SCF complex leads to inhibition of ubiquitination and proteasomal degradation of p53 protein in a ß-TrcP-dependent manner. Successful interaction of SCF(ß-TrcP) complex with ß-TrcP binding motif (DS(52)GNES(56)) present in Vpu is essential because mutant Vpu possessing specific alanine substitutions (DA(52)GNEA(56)) in the ß-TrcP binding motif not only failed to stabilize p53 protein but was also unable to inhibit ubiquitination of p53 protein. Furthermore, Vpu competes efficiently with the interaction of p53 protein with the ß-TrcP subunit of the SCF complex and inhibits subsequent ubiquitination of p53 proteins in a dose-dependent manner. We also observed potent apoptotic activity in a p53 null cell line (H-1299) that was cotransfected with p53 and Vpu-expressing plasmids. Furthermore, MOLT-3 (human T-lymphoblast) cells when infected with vesicular stomatitis virus glycoprotein-pseudotypic HIV-1 possessing wild-type vpu gene exhibited maximum activation of p53/Bax proteins and p53-mediated cell death. These findings establish a novel function of Vpu in modulating the stability of p53 protein that correlates positively with apoptosis during late stages of HIV-1 infection.

Gene expression profiles of HIV-1-infected glia and brain: toward better understanding of the role of astrocytes in HIV-1-associated neurocognitive disorders.

Astrocytes are the major cellular component of the central nervous system (CNS), and they play multiple roles in brain development, normal brain function, and CNS responses to pathogens and injury. The functional versatility of astrocytes is linked to their ability to respond to a wide array of biological stimuli through finely orchestrated changes in cellular gene expression. Dysregulation of gene expression programs, generally by chronic exposure to pathogenic stimuli, may lead to dysfunction of astrocytes and contribute to neuropathogenesis. Here, we review studies that employ functional genomics to characterize the effects of HIV-1 and viral pathogenic proteins on cellular gene expression in astrocytes in vitro. We also present the first microarray analysis of primary mouse astrocytes exposed to HIV-1 in culture. In spite of different experimental conditions and microarray platforms used, comparison of the astrocyte array data sets reveals several common gene-regulatory changes that may underlie responses of these cells to HIV-1 and its proteins. We also compared the transcriptional profiles of astrocytes with those obtained in analyses of brain tissues of patients with HIV-1 dementia and macaques infected with simian immunodeficiency virus (SIV). Notably, many of the gene characteristics of responses to HIV-1 in cultured astrocytes were also altered in HIV-1 or SIV-infected brains. Functional genomics, in conjunction with other approaches, may help clarify the role of astrocytes in HIV-1 neuropathogenesis.

HIV-1 Vpr inhibits cytokinesis in human proximal tubule cells.

Transgenic mouse models of HIV-associated nephropathy (HIVAN) show that expression of HIV-1 genes in kidney cells produces collapsing focal segmental glomerulosclerosis and microcystic tubular disease typical of the human disease. HIV-1 vpr plays an important role in the glomerulosclerosis of HIVAN, especially when it is associated with nef expression in podocytes. Further, Vpr is reported to exacerbate tubular pathology. Here we determined effects of vpr expression on renal tubular epithelial cell function by transducing them with a pseudotyped lentivirus vector carrying HIV-1 vpr and control genes. Vpr expression in the cultured cells impaired cytokinesis causing cell enlargement and multinucleation. This profound in vitro phenotype caused us to reexamine the HIVAN mouse model and human HIVAN biopsies to see if similar changes occur in vivo. Both showed abundant hypertrophic tubule cells similar to the in vitro finding that represents a previously unappreciated aspect of the human disease. Additionally, multinucleated tubular cells were identified in the murine HIVAN model and increased chromosome number was detected in tubular cells of human HIVAN biopsies. Our study provides evidence of a new clinical phenotype in HIVAN that may result from the ability of Vpr to impair cytokinesis.

Gene expression profile of peripheral blood mononuclear cells in response to HIV-VLPs stimulation.

BACKGROUND: Baculovirus-expressed HIV-1 Pr55gag Virus-Like Particles (HIV-VLPs) induce maturation and activation of monocyte-derived dendritic cells (MDDCs) with a production of Th1- and Th2-specific cytokines. RESULTS: The analysis of genomic transcriptional profile of MDDCs, obtained from normal healthy donors and activated by HIV-VLPs, show the modulation of genes involved in the morphological and functional changes characterizing the MDDCs activation and maturation. Similar data are obtained using peripheral blood mononuclear cells (PBMCs), without further selection, showing the feasibility of a direct and "simplified" experimental procedure. CONCLUSIONS: The results here described show that the maturation pattern induced by HIV-VLPs in ex vivo generated MDDCs, can be observed also in CD14-expressing freshly derived PBMCs, with the possible identification of genetic predictors of individual response to immunogens.