Sodium lauryl sulfate abrogates human immunodeficiency virus infectivity by affecting viral attachment.

The microbicidal activity of sodium lauryl sulfate (SLS) against human immunodeficiency virus type 1 (HIV-1) was studied in cultured cells. Pretreatment of HIV-1(NL4-3) with SLS decreased, in a concentration-dependent manner, its infectivity when using 1G5 as target cells. In the absence of a viral pretreatment period or when 1G5 cells were pretreated with SLS, the surfactant-induced inactivation of viral infectivity was less pronounced, especially at concentrations between 375 and 550 microM. SLS had no effect on HIV-1 when the virus was adsorbed to 1G5 cells by a 2-h incubation period. SLS almost completely inhibited the fusion process by decreasing the attachment of HIV-1 to target cells. SLS also inhibited the infectivity of HIV-1-based luciferase reporter viruses pseudotyped with the amphotropic murine leukemia virus envelope (which enters cells in a CD4-, CCR5-, and CXCR4-independent manner), indicating that SLS may inactivate other envelope viruses. In contrast, no effect was seen with vesicular stomatitis virus envelope glycoprotein G (which enters cells through receptor-mediated endocytosis) pretreated with up to 700 microM SLS. SLS also decreased, in a dose-dependent manner, the HIV-1-dependent syncytium formation between 1G5 and J1.1 cells after a 24-h incubation. The reduction of luciferase activity was more pronounced when J1.1 cells (which express HIV-1 proteins on their surface) were pretreated with SLS rather than 1G5 cells. Taken together, our results suggest that SLS could represent a candidate of choice for use in vaginal microbicides to prevent the sexual transmission of HIV and possibly other pathogens causing sexually transmitted diseases.

Highly reliable heterologous system for evaluating resistance of clinical herpes simplex virus isolates to nucleoside analogues.

Clinical resistance of herpes simplex virus (HSV) types 1 and 2 to acyclovir (ACV) is usually caused by the presence of point mutations within the coding region of the viral thymidine kinase (TK) gene. The distinction between viral TK mutations involved in ACV resistance or part of viral polymorphism can be difficult to evaluate with current methodologies based on transfection and homologous recombination. We have developed and validated a new heterologous system based on the expression of the viral TK gene by the protozoan parasite Leishmania, normally devoid of TK activity. The viral TK genes from 5 ACV-susceptible and 13 ACV-resistant clinical HSV isolates and from the reference strains MS2 (type 2) and KOS (type 1) were transfected as part of an episomal expression vector in Leishmania. The susceptibility of TK-recombinant parasites to ganciclovir (GCV), a closely related nucleoside analogue, was evaluated by a simple measurement of the absorbance of Leishmania cultures grown in the presence of the drug. Expression of the TK gene from ACV-susceptible clinical isolates resulted in Leishmania susceptibility to GCV, whereas expression of a TK gene with frameshift mutations or nucleotide substitutions from ACV-resistant isolates gave rise to parasites with high levels of GCV resistance. The expression of the HSV TK gene in Leishmania provides an easy, reliable, and sensitive assay for evaluating HSV susceptibility to nucleoside analogues and for assessing the role of specific viral TK mutations.

Sterically stabilized liposomes bearing anti-HLA-DR antibodies for targeting the primary cellular reservoirs of HIV-1.

The ability of liposomes bearing anti-HLA-DR Fab' fragments at the end termini of polyethyleneglycol chains (sterically stabilized immunoliposomes) to target HLA-DR expressing cells and increase the accumulation of liposomes into lymphoid organs has been evaluated and compared to that of conventional liposomes, sterically stabilized liposomes and conventional immunoliposomes after a single subcutaneous injection to mice. The accumulation of sterically stabilized liposomes in lymph nodes was higher than that of conventional liposomes. Sterically stabilized immunoliposomes accumulated much better than conventional immunoliposomes in all tissues indicating that the presence of PEG has an important effect on the uptake of immunoliposomes by the lymphatic system. Fluorescence microscopy studies showed that sterically stabilized liposomes are mainly localized in macrophage-rich areas such as the subcapsular region of lymph nodes and in the red pulp and marginal zone of the spleen. In contrast, sterically stabilized immunoliposomes mostly accumulated in the cortex in which follicles are located and in the white pulp of the spleen. As the human HLA-DR determinant of the major histocompatibility complex class II is expressed on activated CD4+ T lymphocytes and antigen presenting cells such as monocyte/macrophages and dendritic cells, known as the cellular reservoirs of HIV-1, liposomes bearing anti-HLA-DR antibodies constitute an attractive approach to concentrate drugs in HIV-1 reservoirs and improve their therapeutic effect.

Targeting cell-free HIV and virally-infected cells with anti-HLA-DR immunoliposomes containing amphotericin B.

OBJECTIVE: To evaluate the ability of liposomes bearing anti-HLA-DR Fab' fragments (immunoliposomes) and containing amphotericin B (AmB) to target and neutralize cell-free HIV-1 particles and virally-infected cells. METHODS: The effect of AmB on the attachment and fusion of HIV-1(NL4-3) to Jurkat E6.1 cells has been evaluated using a p24 enzymatic assay. The ability of AmB to inhibit HIV-1-based luciferase reporter viruses pseudotyped with HXB2, AML-V and VSV-G envelopes has been evaluated in Jurkat E6.1 cells. The efficacy of free and immunoliposomal AmB to inhibit cell-free HIV, that have incorporated or not HLA-DR molecules, has been evaluated in HLA-DR/negative (NEG) 1G5 T cells and HLA-DR/positive (POS) Mono Mac 1 cells. RESULTS: AmB inhibited HIV infectivity independently of the nature of viral envelope proteins. Pretreatment of HIV with AmB had no major effect on viral attachment and fusion process to Jurkat E6.1 cells. Immunoliposomal AmB (0.5 microg/ml) led to a 77% inhibition of replication of HLA-DR/POS HIV-1 with no cell toxicity, whereas free AmB had no significant antiviral activity at this concentration. A complete inhibition of viral replication was observed following incubation of viruses with immunoliposomal AmB (2.5 microg/ml). Anti-HLA-DR immunoliposomes containing AmB had no effect on the infectivity of HLA-DR/NEG HIV-1 particles in HLA-DR/NEG T lymphoid cells but completely inhibited replication of viruses in an HLA-DR/POS monocytic cell line. CONCLUSION: The incorporation of neutralizing agents in anti-HLA-DR immunoliposomes could represent a novel therapeutic strategy to specifically target cell-free HIV particles and virally-infected cells to treat HIV infection more efficiently.

In vitro and in vivo evaluations of sodium lauryl sulfate and dextran sulfate as microbicides against herpes simplex and human immunodeficiency viruses.

The efficacy of sodium lauryl sulfate (SLS), a sulfated anionic chaotropic surfactant, and dextran sulfate (DS), a polysulfated carbohydrate, against herpes simplex virus (HSV) and human immunodeficiency virus (HIV) infections was evaluated in cultured cells and in different murine models of HSV infection. Results showed that both SLS and DS were potent inhibitors of the infectivities of various HSV-1 and HSV-2 strains. Pretreatment of HIV-1 (strain NL4-3) with SLS also reduced its infectivity to 1G5 cells. DS prevented the binding of HSV to cell surface receptors and therefore its entry into cells. Pretreatment of HSV-1 (strain F) with 50 microM SLS resulted in a complete loss of virus infectivity to Vero cells. However, viruses were able to enter into cells and to produce in the nuclei capsid shells devoid of a DNA core. The amount of the glycoprotein D gene produced in these cells remained unchanged compared to controls, suggesting that SLS could interfere with the maturation of the virus. At a higher SLS concentration (100 microM), HSV was highly damaged by SLS pretreatment and only a few viral particles could enter into cells to produce abnormal capsids. Although DS was a more potent inhibitor of HSV infectivity in vitro, it was unable to provide any protection in murine models of HSV infection. However, SLS conferred a complete protection of animals infected cutaneously with pretreated viruses. In addition, skin pretreatment of mice with a polymer formulation containing SLS completely prevented the development of cutaneous lesions. More interestingly, intravaginal pretreatment of mice with SLS in a buffered solution also completely protected against lethal HSV-2 infection. Taken together, our results suggest that SLS could thus represent a candidate of choice as a microbicide to prevent the sexual transmission of HIV, HSV, and possibly other pathogens that cause sexually transmitted diseases.

Targeting lymph nodes with liposomes bearing anti-HLA-DR Fab' fragments.

The ability of liposomes bearing anti-HLA-DR Fab' fragments to target cells expressing the human HLA-DR determinant of the major histocompatibility complex class II (MHC-II) has been evaluated and compared to that of conventional liposomes. Anti-HLA-DR immunoliposomes did not bind to HLA-DR-negative cells. In contrast, a high level of binding was observed following incubation of immunoliposomes with cells bearing important levels of human HLA-DR. The accumulation of conventional and murine anti-HLA-DR immunoliposomes in different tissues has been investigated following a single subcutaneous injection given in the upper back of C3H mice. Anti-HLA-DR immunoliposomes resulted in a much better accumulation in the cervical and brachial lymph nodes when compared to conventional liposomes. The accumulation in the liver was similar for both liposomal preparations, whereas an approximately twofold decrease in accumulation was observed for immunoliposomes in the spleen. Given that HLA-DR surface marker is expressed on monocyte/macrophages and activated CD4+ T lymphocytes, the primary cellular reservoirs of the human immunodeficiency virus (HIV), the use of liposomes bearing surface-attached anti-HLA-DR could constitute a convenient strategy to more efficiently treat this debilitating retroviral disease. Moreover, the reported incorporation of high amounts of host-encoded HLA-DR proteins by HIV particles renders the use of liposomes bearing anti-HLA-DR antibodies even more attractive.