Jacob Samson Barnor

Lentiviral-mediated delivery of combined HIV-1 decoy TAR and Vif siRNA as a single RNA molecule that cleaves to inhibit HIV-1 in transduced cells.

RNA interference (RNAi) silences gene expression via short interfering 21–23 mer double-stranded RNA (siRNA) segments that guide cognate mRNA degradation in a sequence-specific manner. On the other hand, HIV-1 decoy TAR RNA are known to competitively interact with the HIV-1 Tat protein, to downregulate the enhanced gene expression from the long terminal repeat (LTR) promoters. Here we report that a novel expression construct, encoding both HIV-1 decoy TAR and Vif siRNA, as a single RNA substrate, was expressed under the control of the human U6 promoter, and later the TAR and siRNA were cleaved into their respective separate RNA by the endogenous RNase III-like enzyme. Each of the cleaved HIV-1 anti-genes then synergistically contributed toward enhancing the inhibition efficacy (> 80%) of HIV-1 replication in transduced Jurkat cells. These results suggest that targeting HIV-1 mRNA with simultaneously expressed intracellular decoy TAR and Vif-siRNA could lead to an effective gene therapy strategy for the control and management of HIV-AIDS.

Hospital-Based Surveillance for Viral Hemorrhagic Fevers and Hepatitides in Ghana

Background Viral hemorrhagic fevers (VHF) are acute diseases associated with bleeding, organ failure, and shock. VHF may hardly be distinguished clinically from other diseases in the African hospital, including viral hepatitis. This study was conducted to determine if VHF and viral hepatitis contribute to hospital morbidity in the Central and Northern parts of Ghana. Methodology/Principal Findings From 2009 to 2011, blood samples of 258 patients with VHF symptoms were collected at 18 hospitals in Ashanti, Brong-Ahafo, Northern, Upper West, and Upper East regions. Patients were tested by PCR for Lassa, Rift Valley, Crimean-Congo, Ebola/Marburg, and yellow fever viruses; hepatitis A (HAV), B (HBV), C (HCV), and E (HEV) viruses; and by ELISA for serological hepatitis markers. None of the patients tested positive for VHF. However, 21 (8.1%) showed anti-HBc IgM plus HBV DNA and/or HBsAg; 37 (14%) showed HBsAg and HBV DNA without anti-HBc IgM; 26 (10%) showed anti-HAV IgM and/or HAV RNA; and 20 (7.8%) were HCV RNA-positive. None was positive for HEV RNA or anti-HEV IgM plus IgG. Viral genotypes were determined as HAV-IB, HBV-A and E, and HCV-1, 2, and 4. Conclusions/Significance VHFs do not cause significant hospital morbidity in the study area. However, the incidence of acute hepatitis A and B, and hepatitis B and C with active virus replication is high. These infections may mimic VHF and need to be considered if VHF is suspected. The data may help decision makers to allocate resources and focus surveillance systems on the diseases of relevance in Ghana.

Procyanidin trimer C1 derived from Theobroma cacao reactivates latent human immunodeficiency virus type 1 provirus

Despite remarkable advances in combination antiretroviral therapy (cART), human immunodeficiency virus type 1 (HIV-1) infection remains incurable due to the incomplete elimination of the replication-competent virus, which persists in latent reservoirs. Strategies for targeting HIV reservoirs for eradication that involves reactivation of latent proviruses while protecting uninfected cells by cART are urgently needed for cure of HIV infection. We screened medicinal plant extracts for compounds that could reactivate the latent HIV-1 provirus and identified a procyanidin trimer C1 derived from Theobroma cacao as a potent activator of the provirus in human T cells latently infected with HIV-1. This reactivation largely depends on the NF-κB and MAPK signaling pathways because either overexpression of a super-repressor form of IκBα or pretreatment with a MEK inhibitor U0126 diminished provirus reactivation by C1. A pan-PKC inhibitor significantly blocked the phorbol ester-induced but not the C1-induced HIV-1 reactivation. Although C1-induced viral gene expression persisted for as long as 48 h post-stimulation, NF-κB-dependent transcription peaked at 12 h post-stimulation and then quickly declined, suggesting Tat-mediated self-sustainment of HIV-1 expression. These results suggest that procyanidin C1 trimer is a potential compound for reactivation of latent HIV-1 reservoirs.

In vitro and in vivo transport and delivery of phosphorothioate oligonucleotides with cationic liposomes.

A recent strategy in gene therapy has been using antiviral genes that are delivered to uninfected cells, either as RNA or DNA, to provide intracellular protection from human immunodeficiency virus type-1 (HIV-1) infection. Antisense oligonucleotides that are complementary to specific target genes suppress gene expression. A variety of techniques are available to enhance the cellular uptake and pharmacological effectiveness of anti-sense oligonucleotides, both in vitro and in vivo. We investigated the intracellular and tissue uptake of an oligonucleotide/cationic lipid complex, using a fluorescently labeled oligonucleotide. The antisense oligonucleotide was designed against the HIV-1 gag gene sequence. A T-cell line (MT-4) and PHA-stimulated peripheral blood mononuclear cells (PBMCs) were both infected with HIV-1NL432 at an MOI of 0.01. One h later, both cultures were washed and treated with medium containing 1 μM antisense oligonucleotide. After a 3-day interval, the HIV-1 antigen expression was monitored by an indirect immunofluorescence assay. At 3 days post-infection, we confirmed that p24 antigen production was inhibited by the anti-sense oligonucleotide/cationic lipid complex at a 1/10 ratio in the PBMCs, using enzyme-linked immunosorbent assay (ELISA). We also confirmed the intracellular existence of the complex by fluorescent microscopy. We investigated different means of transporting the antisense oligonucleotide/cationic lipid complex to mouse tissues by intravenous, intraperitoneal and subcutaneous injections. We observed that the anti-HIV-1 activity of the antisense oligonucleotide/cationic lipid complex was the result of enhanced cellular uptake, both in vitro and in vivo. Therefore, the antisense oligonucleotide/ cationic lipid complex is an excellent system for the transport and delivery of genes to target cells, as it is effective both in vitro and in vivo.

The Middle to 3′ End of the Hiv-1 Vif Gene Sequence is Important for Vif Biological Activity and Could be Used for Antisense Oligonucleotide Targets

The human immunodeficiency virus type-1 (HIV-1)-encoded Vif protein is essential for viral replication, virion production, and pathogenicity. HIV-1 Vif interacts with the endogenous human APOBEC3G protein (an mRNA editor) in target cells to prevent its encapsidation into virions. Some studies have established targets within the HIV-1 vif gene that are important for its biologic function; however, it is important to determine effective therapeutic targets in vif because of its critical role in HIV-1 infectivity and pathogenicity. The present study demonstrates that virions generated in transfected HeLa-CD4+ cells, especially from HIV-1 vif frame-shift mutant (3′-Δvif; 5561-5849), were affected in splicing and had low infectivity in MT-4 cells. In addition, HIV-1 vif antisense RNA fragments constructed within the same region, notably the region spanning nucleic acid positions 5561-5705 (M-3′-AS), which corresponds to amino acid residues 96–144, significantly inhibited HIV-1 replication in MT-4 and reduced the HIV-1 vif mRNA transcripts and reporter gene (EGFP) expression. The generated virions showed low secondary infection in H9 cells. These data therefore suggest that the middle to the 3′ end of vif is important for its biological activity in the target cells.