Werkingsmechanisme en ontwikkeling van resistentie tegen HIV inhibitoren die het binnendringen van het virus verhinderen

Since the discovery of the Human Immunodeficiency Virus (HIV) almost 30years ago, more than 25 million people have been killed by this virus. The existing HIV drug treatment can control, but is not able to cure HIVinfection. The HIV epidemic continues largely unabated with approximately 8000 new infections each day. The proportion of women infected with HIV-1 is increasing, especially in the developing world and the need for new HIV prevention methods that women themselves can initiate and control is indispensable. Microbicides are substances that are intended to reduce or prevent sexual transmission of HIV-1 and other sexually transmitted diseases (STDs) when applied topically inside the vagina or rectum. In the last decade, there has been a shift from the development of broad-spectrum microbicide products with relative non-specific mechanisms of antiviral action, such as surfactants, to antiretroviral microbicides that target specific steps in the viral life cycle.This project was aimed to investigate the precise mode of action and the development of resistance of HIV-1 inhibitors that target early stagesof infection. The first step in the replication cycle of HIV-1 is the adsorption of a virion to the membrane of a CD4+ target cell. The initialinteraction between the viral glycoprotein gp120 and the cellular CD4 receptor introduces conformational changes in gp120 and these result in secondary binding events to the chemokine receptors/HIV co-receptors (CCR5 or CXCR4). Then, the viral glycoprotein gp41 penetrates into the host cell membrane and finally membrane fusion can occur. Each step of this entry process represents an attractive target for the development of potential drugs. In this project we focus on compounds that mainly interact with the viral gp120 and with the cellular receptors: carbohydrate-binding agents (CBAs) that target the glycoproteins on the HIV-1 envelope [e.g. the prokaryotic lectins CV-N and MVN, several plant lectins HHA,GNA and UDA and the monoclonal antibody (mAb) 2G12], polyanions that bind to the HIV-1 envelope (e.g. PRO 2000), CCR5 antagonists (e.g. maraviroc) and CXCR4 antagonists (e.g. AMD3100) that target the two main HIV co-receptors.The chemokine receptor CXCR4 is present in numerous cell-types in different conformations and this could be due to changes in N-glycosylation processes. CXCR4 has two known glycosylation sites and by transfecting U87.CD4 cells with mutated CXCR4 receptors we demonstrated that the glycans on CXCR4 had no direct influence on the infectivity of X4 viruses, andthat various evaluated compounds and lectins (e.g. AMD3100, HHA, GNA and UDA) preserved their antiviral activity.Unlike the glycans on CXCR4, the glycans on the HIV-1 envelope have a crucial role in the transmission and infectivity of the virus. In our project, the antiviral activity of two carbohydrate-specific agents, 2G12 mAb and microvirin (MVN), was examined in detail, and HIV-1 resistant viruses were selected in vitro. The broadly neutralizing 2G12 mAb binds to several specific glycans on gp120, but it showed only activity against clade A and B HIV-1 clinical isolates and strains. The novel prokaryotic lectin MVN has a broad and potent antiviral activity and was able to profoundly inhibit transmission/infection of free virions and cell-associated viruses. Our selected NL4.3-2G12res virus contained only one N-glycosylation deletion in gp120, while in our NL4.3-MVNres virus four specificglycans were deleted. Cross-resistance studies revealed that the MVNresvirus was also completely resistant to the 2G12 mAb, while the 2G12res virus was still sensitive for MVN. In addition, both resistant viruses kept their sensitivity to CV-N, various plant lectins and other classes of HIV entry inhibitors, and the NL4.3-2G12res virus became even more sensitive to several CBAs.However a microbicide candidate does not only need to have a broad and potent antiviral activity and a high genetic barrier, it also has to be safe and well tolerated when used vaginal or rectal. Previously, the absence of specific safety biomarkers to examine possible side-effects of microbicide candidates has led to the failure of e.g. nonoxynol-9 in phase 3 clinical microbicide trials. Toxic effects were reported and they even caused an increased incidence of HIV transmission and other STDs. Thus, there is an urgent need in identifying and evaluating a new generation of accessible and easy to use agents with a safe and potent antiviral activity. Cyanovirin-N (CV-N), one of the most advanced CBAs in development as a microbicide, was found active in viral transmission models in macaques. However, very little data were available on the possible side-effects of this lectin. Our detailed investigation demonstrated that CV-Nenhanced the expression of the cellular activation markers CD25, CD69 and HLA-DR in PBMCs. In addition, CD4+ T cells pretreated with CV-N became more susceptible for CCR5-tropic (R5) HIV-1 infection, and CV-N dramatically enhanced the production of a wide variety of cytokines. In comparison, MVN, HHA, GNA, UDA, AMD3100 and maraviroc induced markedly less, if any, stimulatory effects. MVN did induce the production of various cytokines in preincubated PBMCs although this effect was less pronounced asfor CV-N.The safety profile of the polyanion PRO 2000 was also evaluated and PRO2000 had minor effects on the expression level of several cellular activation markers and enhanced the production of a small number of cytokines/chemokines in PBMCs. Unfortunately, PRO 2000 recently failed in a phase 3 clinical microbicide trial, but the compound was found to be safe and well tolerated. Previously, the antiviral activity of PRO 2000 was attributed to its binding to HIV-1 gp120 and interference with virus attachment to and/or fusion with CD4+ T cells. Our data demonstrate that PRO 2000 interacts not only with viral gp120 but also with the cellular CD4 and CXCR4 receptors. The observed CXCR4 antagonistic properties of PRO 2000 can be considered as a potential additional mechanism of antiviral action and it is peculiar that one compound can have multiple and different interactions at the viral-cellular membrane level.In this project we studied various classes of antiviral compounds and deciphered further their very often multiple mechanisms of action and viral resistance profile. However, caution should be given for the development of all these compounds as microbicidal agents. The use of CV-N as a safe microbicide raises questions because CV-N has clearly stimulatory/mitogenic activity and induces high amounts of a large number of cytokines. However, other lectins (e.g. MVN, HHA and UDA) exist that have broad and potent antiviral activity and much less, if any, stimulatory properties and they stand out as potential candidates for microbicidal development.status: publishe