Sustained and specific in vitro inhibition of HIV-1 replication by a protease inhibitor encapsulated in gp120-targeted liposomes.

Selective delivery of antiretrovirals to human immunodeficiency virus (HIV) infected cells may reduce toxicities associated with long-term highly active antiretroviral therapy (HAART), may improve therapeutic compliance and delay the emergence of resistance. We developed sterically stabilized pegylated liposomes coated with targeting ligands derived from the Fab' fragment of HIV-gp120-directed monoclonal antibody F105, and evaluated these liposomes as vehicles for targeted delivery of a novel HIV-1 protease inhibitor. We demonstrated that the immunoliposomes were selectively taken up by HIV-1-infected cells and localized intracellularly, enabling the establishment of a cytoplasmic reservoir of protease inhibitor. In antiviral experiments, the drug delivered by the immunoliposomes showed greater and longer antiviral activity than comparable concentrations of free drug or drug encapsulated in non-targeted liposomes. In conclusion, by combining a targeting moiety with drug-loaded liposomes, efficient and specific uptake by non-phagocytic HIV-infected cells was facilitated, resulting in drug delivery to infected cells. This approach to targeted delivery of antiretroviral compounds may enable the design of drug regimens for patients that allow increased therapeutic adherence and less toxic treatment of HIV infection.

Antiviral compounds show enhanced activity in HIV-1 single cycle pseudovirus assays as compared to classical PBMC assays.

HIV-1 Env pseudotyped viruses (PV) are an attractive tool for studying the antiviral activities of compounds interfering with virus entry into a target cell. To investigate whether results obtained in PV assays are relevant biologically, the antiviral activity of 6 reference compounds was compared on 5 virus isolates of different clades using three assays: (1) replicating virus in peripheral blood mononuclear cells (PBMCs), (2) PV in CD4 and CCR5- or CXCR4 co-receptor expressing Ghost cells, and (3) PV in PBMCs. A significant linear relationship was found between both single-cycle PV assays (P<0.0001, R2=0.75). Moreover, both assays showed enhanced sensitivity to the antiretrovirals tested (P=0.013 and 0.015, respectively) as compared to the PBMC assay with replication-competent virus. Most importantly, results from the latter assay could be predicted significantly from both PV assays, in which either Ghost target cells (P<0.0001, R2=0.61) or PBMCs (P<0.0001, R2=0.55) were used. The usefulness of the PV assay was demonstrated further by investigating the impact of the HIV-1 Env subtype on the antiviral activity of five new compounds derived from the entry inhibitor BMS806.

A time-resolved fluorescence assay to identify small-molecule inhibitors of HIV-1 fusion.

Fusion of host cell and human immunodeficiency virus type 1 (HIV-1) membranes is mediated by the 2 "heptad-repeat" regions of the viral gp41 protein. The collapse of the C-terminal heptad-repeat regions into the hydrophobic grooves of a coiled-coil formed by the corresponding homotrimeric N-terminal heptad-repeat regions generates a stable 6-helix bundle. This brings viral and cell membranes together for membrane fusion, facilitating viral entry. The authors developed an assay based on soluble peptides derived from the gp41 N-terminal heptad-repeat region (IQN36) as well as from the C-terminal region (C34). Both peptides were labeled with fluorophores, IQN36 with allophycocyanin (APC) and C34 with the lanthanide europium (Eu3+). Formation of the 6-helix bundle brings both fluorophores in close proximity needed for Förster resonance energy transfer (FRET). Compounds that interfere with binding of C34-Eu with IQN36-APC suppress the FRET signal. The assay was validated with various peptides and small molecules, and quenching issues were addressed. Evaluation of a diversified compound collection in a high-throughput screening campaign enabled identification of small molecules with different chemical scaffolds that inhibit this crucial intermediate in the HIV-1 entry process. This study's observations substantiate the expediency of time-resolved FRET-based assays to identify small-molecule inhibitors of protein-protein interactions.

Binding kinetics, uptake and intracellular accumulation of F105, an anti-gp120 human IgG1kappa monoclonal antibody, in HIV-1 infected cells.

The use of targeting moieties is a new and exciting field of scientific research for facilitating the specific delivery of therapeutic agents in HIV-infected patients. The interaction of a potential targeting moiety with its ligand is a crucial factor in the evaluation of a targeted approach for chemotherapeutic intervention. Therefore, we have further characterized the interaction between a potential targeting agent, the monoclonal human antibody F105, and its ligand gp120, a glycoprotein expressed on the surface of HIV-1 infected cells. We demonstrate the specificity of binding and entry of F105 to infected cells. F105 was rapidly taken up into the cell and accumulated in the Golgi apparatus. Kinetic analysis of the F105-gp120 interaction revealed an equilibrium dissociation constant (K(D)) of 0.62 nM, compared with the gp120-CD4 interaction where the K(D) was determined at 35 nM. Consequently, F105 displayed a higher gp120 affinity. This was due to a slower dissociation as compared with the natural ligand. These data further underline the potential of monoclonal antibodies as targeting agents, and offer new insights into the possibility of F105 as a targeting moiety for the delivery of antiretroviral drugs to HIV-1 infected cells.

Development of models and detection methods for different forms of cytomegalovirus for the evaluation of viral inactivation agents.

BACKGROUND: Cytomegalovirus (CMV) is transmitted by transfusion of infected blood products and can cause serious diseases in specific risk groups. CMV can be present in infected blood as cell-free virus (CFV), cell-associated actively replicating virus (CAV), and cell-associated latent virus (LV). STUDY DESIGN AND METHODS: In vitro models for all three infectious forms of CMV and virus detection assays based on both tissue culture and polymerase chain reaction (PCR) were developed. The utility of the CMV model systems and assays were tested by validation studies of a novel pathogen inactivation agent, PEN110, for red blood cells. RESULTS: Reproducible high titers of CFV and CAV were obtained by optimized tissue culture techniques for CMV-infected MRC-5 cells. An LV model was obtained with CMV-infected THP-1 cells and reactivation of virus replication by phorbol ester treatment. The model systems showed that PEN110 treatment is effective against all three forms of CMV as measured by tissue culture-based infectivity assays and a long-range PCR method specific for detection of damage to CMV viral DNA. CONCLUSION: This study describes model systems to the relevant forms of CMV in blood and detection assays that can be used to evaluate the efficacy of viral inactivation agents.

Development of a homogeneous screening assay for automated detection of antiviral agents active against severe acute respiratory syndrome-associated coronavirus.

The severity and global spread of the 2003 outbreak of the severe acute respiratory syndrome-associated coronavirus (SARS-CoV) highlighted the risks to human health posed by emerging viral diseases and emphasized the need for specific therapeutic agents instead of relying on existing broadly active antiviral compounds. The development of rapid screening assays is essential for antiviral drug discovery. Thus, a screening system for anti-SARS-CoV agents was developed, which evaluated compound potency, specificity and cytotoxicity at the initial screening phase. Cell lines were engineered to constitutively express an enhanced green fluorescent protein (EGFP) and used to detect (1) antiviral potency in SARS-CoV infection tests; (2) antiviral specificity in tests using the porcine coronavirus transmissible gastroenteritis virus (TGEV); and (3) cytotoxicity in the same assays without virus challenge. The assay system involves minimal manipulation after assay set-up, facilitates automated read-out and minimizes risks associated with hazardous viruses. The suitability of this assay system in drug discovery was demonstrated by screening of 3388 small molecule compounds. The results show that these assays can be applied to high-throughput screening for identification of inhibitors selectively active against SARS-CoV.

Development of a sensitive PCR inhibition method to demonstrate HBV nucleic acid inactivation.

BACKGROUND: The evaluation of pathogen reduction technologies with relevant viruses currently contaminating the blood supply is limited by the availability of high-titer virus inocula and sensitive in vitro or in vivo infectivity assays. Because HBV infectivity can only be assessed by in vivo studies with chimpanzees, a sensitive PCR inhibition assay was developed to measure PEN110 inactivation of HBV. STUDY DESIGN AND METHODS: PCR amplification of 1.1 kb of HBV genome was optimized to determine DNA damage introduced by treatment with PEN110 in RBCs. Inactivation of duck HBV (DHBV) in RBCs, with measurement of the in vitro infectivity, was performed to validate the PCR assay. RESULTS: The PCR was highly specific and sensitive for amplification of the HBV genome and used to demonstrate a reduction of at least 7.2 and 8.1 log geq per mL within the first 18 hours of PEN110 treatment. PEN110 inactivation of DHBV was also achieved within the first 18 hours with a reduction factor of at least 5.0 log tissue culture infectious dose 50 percent per mL, suggesting that PCR inhibition is an alternative to infectivity assays. CONCLUSION: This study establishes PCR inhibition as a reasonable approach to assess the efficiency of PEN110 inactivation of human pathogens with human plasma donations that have been found to contain high titers of relevant agents during different stages of infection.

Genetic and functional analysis of full-length human immunodeficiency virus type 1 env genes derived from brain and blood of patients with AIDS.

The genetic evolution of human immunodeficiency virus type 1 (HIV-1) in the brain is distinct from that in lymphoid tissues, indicating tissue-specific compartmentalization of the virus. Few primary HIV-1 envelope glycoproteins (Envs) from uncultured brain tissues have been biologically well characterized. In this study, we analyzed 37 full-length env genes from uncultured brain biopsy and blood samples from four patients with AIDS. Phylogenetic analysis of intrapatient sequence sets showed distinct clustering of brain relative to blood env sequences. However, no brain-specific signature sequence was identified. Furthermore, there was no significant difference in the number or positions of N-linked glycosylation sites between brain and blood env sequences. The patterns of coreceptor usage were heterogeneous, with no clear distinction between brain and blood env clones. Nine Envs used CCR5 as a coreceptor, one used CXCR4, and two used both CCR5 and CXCR4 in cell-to-cell fusion assays. Eight Envs could also use CCR3, CCR8, GPR15, STRL33, Apj, and/or GPR1, but these coreceptors did not play a major role in virus entry into microglia. Recognition of epitopes by the 2F5, T30, AG10H9, F105, 17b, and C11 monoclonal antibodies varied among env clones, reflecting genetic and conformational heterogeneity. Envs from two patients contained 28 to 32 N-glycosylation sites in gp120, compared to around 25 in lab strains and well-characterized primary isolates. These results suggest that HIV-1 Envs in brain cannot be distinguished from those in blood on the basis of coreceptor usage or the number or positions of N-glycosylation sites, indicating that other properties underlie neurotropism. The study also demonstrates characteristics of primary HIV-1 Envs from uncultured tissues and implies that Env variants that are glycosylated more extensively than lab strains and well-characterized primary isolates should be considered during development of vaccines and neutralizing antibodies.

West Nile virus in blood: stability, distribution, and susceptibility to PEN110 inactivation.

BACKGROUND: The outbreak of West Nile virus (WNV) is the most recent reminder that the blood supply continues to be vulnerable to emerging and reemerging pathogens. A potentially prospective approach to reducing the risk of transfusion-transmitted infections of a known or newly emerging microbe is implementation of a broad-spectrum pathogen reduction technology. The purpose of this study was to evaluate the susceptibility of WNV to PEN110 inactivation in RBCs and to characterize the WNV interaction with blood, including the stability of WNV in RBCs stored at 1 to 6 degrees C, its distribution and infectivity, and its ability to infect WBCs. STUDY DESIGN AND METHODS: Inactivation was performed with three WNV isolates spiked into WBC-reduced RBCs. The stability of the virus was evaluated by spiking two viral loads into RBCs followed by storing at 1 to 6 degrees C for up to 42 days. The distribution of the virus in plasma, RBCs, and PBMCs was evaluated with whole blood from infected hamsters. Finally, in vitro propagation of WNV was evaluated with the THP-1 cell line and primary monocytes. RESULTS: The kinetics of PEN110 inactivation of WNV isolates RI-44, NJ-176, and 99-3494031 were fast and complete within 24 hours with reduction factors of 5 to 7 log plaque-forming units per mL. WNV remained infectious for up to 42 days at 1 to 6 degrees C. The WNV titers in whole blood, plasma, RBCs, and PBMC fractions were equally distributed and ranged from 2 to 3 log tissue culture infectious dose 50 percent per mL. Productive infection of stimulated monocytes and THP-1 cells was also demonstrated. CONCLUSIONS: These studies demonstrated that PEN110 efficiently inactivated WNV in RBCs and whole blood from infected hamsters to the limit of detection. WNV survived in RBCs stored at 1 to 6 degrees C with a gradual loss of titer but infectivity could still be observed for up to 42 days. In addition, it was observed that WNV was equally distributed in all blood fractions including PBMCs and it was possible to establish productive infection of a human monocytic cell line and stimulated human monocytes.

Inactivation of HIV in blood.

BACKGROUND: The residual risk of HIV infection after HIV screening tests in combination with the risk of new emerging pathogens entering the blood supply has sparked research on the development of a technology for reduction of pathogens in RBCs. STUDY DESIGN AND METHODS: HIV-1 was treated with PEN110 (INACTINE) and analyzed for the kinetics of virus reduction in RBC, the effect of PEN110 on nucleic acids, the integrity of the virus morphology and viral proteins, and the ability of the virus to bind HIV cell receptors and enter susceptible cells. RESULTS: PEN110 effectively reduced HIV-1 to the limit of detection for a reduction factor of at least 5.57 log 50 percent tissue culture infectious dose per bulk test. The PEN110-treated virions maintained their morphology, protein integrity, and functionality. However, the PEN110-treated HIV-1 RNA genome was neither functional to serve as a template for RT-PCR amplification of about 1 kb nor able to support viral DNA synthesis in cell culture. CONCLUSION: These results suggest that PEN110 inactivates HIV-1 by targeting the viral nucleic acid.