Development and Evaluation of an Enzyme-Linked Immunosorbent Assay for Dengue Capsid

The astonishing speed with which Dengue has spread across the world and the severity of its infection make Dengue a prime threat to human life worldwide. Unfortunately, to date there are no effective vaccines or treatments against Dengue. Since only a few assays permit rapid and sensitive detection of Dengue, we developed a specific antigen capture enzyme-linked immunosorbent assay (ELISA) for the abundant structural Dengue-2 capsid protein. We showed that the ELISA allows rapid and sensitive detection of Dengue-2 replication in various cell lines including human and mosquito cells. Using anti-capsid antibodies, we demonstrated that the capsid ELISA is as accurate as other well-characterized Dengue assays such as intracellular FACS staining (IFSA) and fluorescent focus (FFA) assays. The capsid ELISA not only represents a useful tool for in vitro basic research, but it may also represent a valuable diagnostic tool for Dengue infection in patients

Inhibition of HIV Env binding to cellular receptors by monoclonal antibody 2G12 as probed by Fc-tagged gp120

During natural HIV infection, an array of host receptors are thought to influence virus attachment and the kinetics of infection. In this study, to probe the interactions of HIV envelope (Env) with various receptors, we assessed the inhibitory properties of various anti-Env monoclonal antibodies (mAbs) in binding assays. To assist in detecting Env in attachment assays, we generated Fc fusions of full-length wild-type gp120 and several variable loop-deleted gp120s. Through investigation of the inhibition of Env binding to cell lines expressing CD4, CCR5, DC-SIGN, syndecans or combinations thereof, we found that the broadly neutralizing mAb, 2G12, directed to a unique carbohydrate epitope of gp120, inhibited Env-CCR5 binding, partially inhibited Env-DC-SIGN binding, but had no effect on Env-syndecan association. Furthermore, 2G12 inhibited Env attachment to primary monocyte-derived dendritic cells, that expressed CD4 and CCR5 primary HIV receptors, as well as DC-SIGN, and suggested that the dual activities of 2G12 could be valuable in vivo for inhibiting initial virus dissemination and propagation

Bioengineering and Semisynthesis of an Optimized Cyclophilin Inhibitor for Treatment of Chronic Viral Infection.

Inhibition of host-encoded targets, such as the cyclophilins, provides an opportunity to generate potent high barrier to resistance antivirals for the treatment of a broad range of viral diseases. However, many host-targeted agents are natural products, which can be difficult to optimize using synthetic chemistry alone. We describe the orthogonal combination of bioengineering and semisynthetic chemistry to optimize the drug-like properties of sanglifehrin A, a known cyclophilin inhibitor of mixed nonribosomal peptide/polyketide origin, to generate the drug candidate NVP018 (formerly BC556). NVP018 is a potent inhibitor of hepatitis B virus, hepatitis C virus (HCV), and HIV-1 replication, shows minimal inhibition of major drug transporters, and has a high barrier to generation of both HCV and HIV-1 resistance

DEB025 (Alisporivir) Inhibits Hepatitis C Virus Replication by Preventing a Cyclophilin A Induced Cis-Trans Isomerisation in Domain II of NS5A

DEB025/Debio 025 (Alisporivir) is a cyclophilin (Cyp)-binding molecule with potent anti-hepatitis C virus (HCV) activity both in vitro and in vivo. It is currently being evaluated in phase II clinical trials. DEB025 binds to CypA, a peptidyl-prolyl cis-trans isomerase which is a crucial cofactor for HCV replication. Here we report that it was very difficult to select resistant replicons (genotype 1b) to DEB025, requiring an average of 20 weeks (four independent experiments), compared to the typically <2 weeks with protease or polymerase inhibitors. This indicates a high genetic barrier to resistance for DEB025. Mutation D320E in NS5A was the only mutation consistently selected in the replicon genome. This mutation alone conferred a low-level (3.9-fold) resistance. Replacing the NS5A gene (but not the NS5B gene) from the wild type (WT) genome with the corresponding sequence from the DEB025res replicon resulted in transfer of resistance. Cross-resistance with cyclosporine A (CsA) was observed, whereas NS3 protease and NS5B polymerase inhibitors retained WT-activity against DEB025res replicons. Unlike WT, DEB025res replicon replicated efficiently in CypA knock down cells. However, DEB025 disrupted the interaction between CypA and NS5A regardless of whether the NS5A protein was derived from WT or DEB025res replicon. NMR titration experiments with peptides derived from the WT or the DEB025res domain II of NS5A corroborated this observation in a quantitative manner. Interestingly, comparative NMR studies on two 20-mer NS5A peptides that contain D320 or E320 revealed a shift in population between the major and minor conformers. These data suggest that D320E conferred low-level resistance to DEB025 probably by reducing the need for CypA-dependent isomerisation of NS5A. Prolonged DEB025 treatment and multiple genotypic changes may be necessary to generate significant resistance to DEB025, underlying the high barrier to resistance

HSV Neutralization by the Microbicidal Candidate C5A

Genital herpes is a major risk factor in acquiring human immunodeficiency virus type-1 (HIV-1) infection and is caused by both Herpes Simplex virus type 1 (HSV-1) and HSV-2. The amphipathic peptide C5A, derived from the non-structural hepatitis C virus (HCV) protein 5A, was shown to prevent HIV-1 infection but neither influenza nor vesicular stomatitis virus infections. Here we investigated the antiviral function of C5A on HSV infections. C5A efficiently inhibited both HSV-1 and HSV-2 infection in epithelial cells in vitro as well as in an ex vivo epidermal infection model. C5A destabilized the integrity of the viral HSV membrane. Furthermore, drug resistant HSV strains were inhibited by this peptide. Notably, C5A-mediated neutralization of HSV-1 prevented HIV-1 transmission. An in vitro HIV-1 transmigration assay was developed using primary genital epithelial cells and HSV infection increased HIV-1 transmigration. Treatment with C5A abolished HIV-1 transmigration by preventing HSV infection and by preserving the integrity of the genital epithelium that was severely compromised by HSV infection. In conclusion, this study demonstrates that C5A represents a multipurpose microbicide candidate, which neutralizes both HIV-1 and HSV, and which may interfere with HIV-1 transmission through the genital epithelium

Feline Immunodeficiency Virus OrfA Is Distinct from Other Lentivirus Transactivators

The feline immunodeficiency virus (FIV) accessory factor, OrfA, facilitates transactivation of transcription directed by elements of the viral long terminal repeat (LTR). In order to map OrfA domains required for this transactivation, we used N- and C-terminal deletion constructs of the protein, expressed in a Gal4-based transactivation system. The results demonstrated that FIV OrfA, unlike other lentiviral transactivators such as visna virus Tat, is unable to transactivate from minimal promoter-based reporters and requires additional elements of the viral LTR. Stable CrFK-based cell lines were prepared that expressed OrfA to readily detectable levels and in which we were able to demonstrate 32-fold transactivation of an LTR-chloramphenicol acetyltransferase construct. Transactivation was heavily dependent on the presence of an ATF site within the viral LTR. Changing the translation initiation codon context substantially increased the level of production of OrfA from a bicistronic message that also encodes Rev. In the presence of a more favorable context sequence, the upstream expression of OrfA increased 21-fold, with only a 0.5-fold drop in downstream Rev expression. This suggests that Rev translation may occur via an internal ribosomal entry site rather than by leaky scanning

A recombination-efficient baculovirus vector for simultaneous expression of multiple genes

The baculovirus system is an extremely powerful tool for expression of heterologous genes in eukaryotic environment. A multiple expression vector, pBacUCmP3, was constructed which harbored two copies of the Autographa californica nuclear polyhedrosis virus very late gene promoter and the Drosophila melanogaster 70-kDa heat-shock protein (hsp70) promoter with downstream unique restriction sites for cloning of three independent foreign genes. Co-transfection of pBacUCmP3 with Bsu36I-linearized viral DNA yields recombinant progeny viruses at very high frequencies. The utility of this multiple expression transfer vector was demonstrated using three heterologous reporter genes encoding the β-subunit of the human chorionic gonadotropin hormone, firefly luciferase and the bacterial β-galactosidase (βGal) enzyme. The expression of reporter genes, monitored at various times post-infection, confirmed that while β-Gal synthesis was under the transcriptional control of the hsp70 promoter, the βhCG and Luc proteins were synthesized as a function of polyhedrin promoter activation profile. This vector will be useful for multiple synthesis of proteins at different time points

Cyclophilin Inhibitors Remodel the Endoplasmic Reticulum of HCV-Infected Cells in a Unique Pattern Rendering Cells Impervious to a Reinfection.

The mechanisms of action by which cyclophilin inhibitors (CypI) interfere with the HCV life cycle remain poorly understood. We reported that CypI and NS5A inhibitors (NS5Ai), but not other classes of anti-HCV agents, prevent assembly of double membrane vesicles (DMVs), which protect replication complexes. We demonstrated that both NS5A and the isomerase cyclophilin A (CypA) are required for DMV formation. Here, we examined whether CypI mediate an additional antiviral effect that could further explain the high efficacy of CypI. We identified a unique action of CypI. CypI remodel the organization of the endoplasmic reticulum (ER) of HCV-infected cells, but not of uninfected cells. This effect is specific since it was not observed for other classes of anti-HCV agents including NS5Ai, and has no effect on the viability of CypI-treated cells. Since ER serves as platform for the establishment of HCV replication complexes, we asked whether the ER reorganization by CypI would prevent cells from being newly infected. Remarkably, CypI-treated HCV-pre-infected cells remain totally impervious to a reinfection, suggesting that the CypI-mediated ER reorganization prevents a reinfection. This block is not due to residual CypI since CypI-resistant HCV variants also fail to infect these cells. The ER reorganization by CypI is rapid and reversible. This study provides the first evidence that CypI trigger a unique ER reorganization of infected cells, rendering cells transiently impervious to a reinfection. This study further suggests that the HCV-induced ER rearrangement represents a key target for the development of new therapies

Feline immunodeficiency virus OrfA alters gene expression of splicing factors and proteasome-ubiquitination proteins

AbstractExpression of the feline immunodeficiency virus (FIV) accessory protein OrfA (or Orf2) is critical for efficient viral replication in lymphocytes, both in vitro and in vivo. OrfA has been reported to exhibit functions in common with the human immunodeficiency virus (HIV) and simian immunodeficiency virus (SIV) accessory proteins Vpr and Tat, although the function of OrfA has not been fully explained. Here, we use microarray analysis to characterize how OrfA modulates the gene expression profile of T-lymphocytes. The primary IL-2-dependent T-cell line 104-C1 was transduced to express OrfA. Functional expression of OrfA was demonstrated by trans complementation of the OrfA-defective clone, FIV-34TF10. OrfA-expressing cells had a slightly reduced cell proliferation rate but did not exhibit any significant alteration in cell cycle distribution. Reverse-transcribed RNA from cells expressing green fluorescent protein (GFP) or GFP+OrfA were hybridized to Affymetrix HU133 Plus 2.0 microarray chips representing more than 47,000 genome-wide transcripts. By using two statistical approaches, 461 (Rank Products) and 277 (ANOVA) genes were identified as modulated by OrfA expression. The functional relevance of the differentially expressed genes was explored by Ingenuity Pathway Analysis. The analyses revealed alterations in genes critical for RNA post-transcriptional modifications and protein ubiquitination as the two most significant functional outcomes of OrfA expression. In these two groups, several subunits of the spliceosome, cellular splicing factors and family members of the proteasome-ubiquitination system were identified. These findings provide novel information on the versatile function of OrfA during FIV infection and indicate a fine-tuning mechanism of the cellular environment by OrfA to facilitate efficient FIV replication