< Back to previous page
Publication
Anti-HIV activity and mode of action of naturally occurring peptides interfering with the viral entry process
Book - Dissertation
At the end of 2012, approximately 34 million people, including children, were infected worldwide with the human immunodeficiency virus (HIV), the etiological agent of AIDS (Acquired Immune Deficiency Syndrome). Sub-Saharan Africa is the most severly affected region. Especially women are highly susceptible to HIV infection, based on socio-economical and cultural aspects such as religion, polygamy, poverty, rape and war. The transmission rate of HIV infection increases significantly in combination with other genital diseases like bacterial vaginosis, syphilis or genital ulcers caused by herpes simplex virus type 2 (HSV-2) infections. According to UNAIDS, the number of new HIV infections is declining in Sub-Saharan Africa, while the opposite is true in other parts of the world such as Eastern Europe, Middle East and Central Asia. The huge worldwide diversity of HIV subtypes (e.g. the presence of subtype B in Europe and the US and subtype C in Africa) makes it very difficult to develop an effective vaccine. However, the presence of a wide variety of antiretroviral drugs, used in combination therapy, switches HIV from a lethal disease into a chronic disorder. At present, none of the current anti-HIV drugs can eradicate the virus, making HIV treatment a lifelong commitment. Therefore it is necessary to generate also effective prevention campaigns to make people aware of the risks and lifelong consequences of HIV/AIDS infections. These prevention methods in combination with effective treatment options are a crucial step in trying to decrease the number of novel HIV infections. The search for an effective microbicide has been ongoing for more than a decade in order to reduce the sexual transmission of HIV. Microbicides are tools (e.g. vaginal/rectal gels, films, suppositories or intravaginal ring devices) that women, and even (homosexual) men, can use without the knowledge of their partners or other persons. As various clinical trials using broad-spectrum aspecific microbicides (e.g. spermicides, detergents) failed, microbicidal research has focused more on specific antiviral compounds, which act a crucial steps (e.g. reverse transcriptase) in the replication cycle of HIV. This led to a proof-of-concept, whereby a 1% vaginal tenofovir gel, applied within 24 h before and after coitus, reduced the sexual transmission of HIV-1 and HSV-2 in women with, respectively, 39% and 51%. HIV particles have highly glycosylated structures at the surfaces, which are considered as a target for a novel class of antiviral compounds, the carbohydrate-binding agents (or CBAs). Various members of the CBAs were reported to be potential microbicidal candidates. As for the treatment of HIV/AIDS infections, an effective microbicide shall also presumably consist of a combination of at least two different products. Oral tenofovir, the most prescribed anti-HIV drug for the treatment of HIV/AIDS infections, was mid 2012 also approved by the US FDA in combination with emtricitabine as Truvada(R) for pre-exposure prophylaxis (PrEP) against HIV infections and transmission.
Inour research, we showed that tenofovir can be combined with various members of the class of CBAs (e.g. HHA, GNA, MVN, BanLec,...) and that this synergy increases their combined antiviral potency and decreases potential cytotoxicity of these agents (Chapter 3).Despite the fact that the glycans present on gp120 are an attractive target for antiviral therapy, there is a huge diversity between the HIV subtypes as such. We specifically investigated the subtypes B and C, as they account for >60% of the total HIV-1 infections worldwide. In a follow-up study, we investigated if griffithsin (GRFT), the most potent anti-HIV-1 CBA described to date, could be combined with other classes of antiretroviral drugs (e.g. integrase inhibitors, entry inhibitors and reverse transcriptase inhibitors) and if there is a complete loss of antiviral activity when certain sugars differ between the included subtypes. It was reported that the N-linked glycans N295 and N234 on gp120 were involved in the interaction with GRFT and we focused mainly on these two glycans in our experiments. We observed that GRFT lost only minor activity when one or even both sugars were absent. Moreover, GRFT showed synergy with the HIV-1 inhibitors maraviroc (CCR5 antagonist), enfuvirtide (gp41 fusion peptide inhibitor), raltegravir (integrase inhibitor) and tenofovir (reverse transcriptase inhibitor) (Chapter 4). Each CBA binds to specific sugar moieties (e.g. mannose or N-aceylglucosamine) and their bridges (e.g. alpha(1,6) or alpha(1,2) linkages). Based on the above mentioned results, we investigated if CBAs as such could be combined against wild type HIV-1 and HIV-2 strains, and also against certain HIV-1 CBAresistant strains (Chapter 5). We performed most of the experiments with GRFT. First, we could prove that GRFT and the banana lectin (BanLec) also possess an equally potent anti-HIV-2 activity. Various (pre-)clinical antiretroviral agents have a strong reduced, if any, antiviral activity against HIV-2 (e.g. enfuvirtide, microvirin (MVN)). We could also demonstrate that GRFT, in combination with various CBAs (such as MVN, BanLec or the broad neutralizing anti-carbohydrate-binding mAb 2G12) inhibited HIV replication more efficiently. Surprisingly, when the CBAs HHA and GNA were combined, rather antagonistic to additive effects were observed against wild type HIV viruses and synergy against CBAresistant HIV-1 strains, which deleted 1 (as seen with HIV-1 NL4.32G12mAbres. virus) to 4 N-linked glycans (in case of HIV-1 NL4.3MVNres. virus). In addition, GRFT and BanLec kept their potent antiviral activity against these two CBAresistant viruses. These results clearly indicate that (i) CBAs could be combined as such, (ii) that they exhibit different binding patterns on the glycans of gp120 and that (iii) affinity and kinetics are crucial factors in their antiviral activity.Semen and cervicovaginal secretions of HIV-infected persons contain cell-free HIV particles and HIV-infected immune cells (e.g. T cells and macrophages). These cells can easily transmit HIV through T cell - T cell contacts to uninfected CD4+ uninfected T cells. The dendritic cells (DCs) are a population of cells, which detect and capture HIV particles upon infection. These cells express DC-SIGN (dendritic cell-specific intercellular adhesion molecule-3 grabbing non-integrin), an attachment receptor for various pathogens such as HIV. Upon receptor interaction the virus is efficiently transmitted to the non-infected CD4+ T cells in the lymph nodes. So, we investigated in more detail if GRFT could also inhibit, alone and in combination with other classes of antiretroviral drugs (namely entry inhibitors, reverse transcriptase inhibitors, integrase inhibitors and protease inhibitors), the HIV-1 transmission/replication and the CD4+ target T cell destruction as well, through these T cell - T cell and DCs - T cell contacts (Chapter 6). These intercellular contacts result in the formation of multinucleated giant cells or syncytia. We were able to visualize how these giant cells were formed in function of time by the JuLI(TM) live cell-imaging viewer and how the CD4+ target T cell population was destroyed. These data also clearly show that GRFT, alone and in combination, prevents both cell-to-cell HIV-1 transmission and destruction of the CD4+ T cell population.In the second part of this thesis, we focused more on the novel antibiotic peptides feglymycin (FGM) and labyrinthopeptins (LabyA1). We investigated more in depth their antiviral spectrum and mechanism of action. FGM, a peptide of 13 amino acids, showed a consistent broad-spectrum anti-HIV activity in inhibiting infection with cell-free HIV particles as well as in cocultivation assays between persistently HIV-infected T cells and CD4+ non-infected T cells. In the DC-SIGN dependent transmission route FGM inhibits not the capture of the virus on DC-SIGN, but subsequently the viral replication upon cocultivation with the CD4+ target T cells. These data clearly indicate that the glycans present on gp120 are not involved in the anti-HIV mechanism of action of FGM. By the use of various virus binding assays (e.g. surface plasmon resonance, ELISAs, flow cytometry) and viral replication studies, we were able to prove that FGM inhibited the CD4/gp120 binding, through interaction with the viral envelope protein gp120. Alanine scanning mutagenesis studies of FGM, where each amino acid is systematically replaced by a neutral alanine, resulted in a significant loss of antiviral activity and affinity for gp120, when the L-aspartate at position 13 was replaced by a neutral alanine. Cultivating HIV-1 in the presence of increasing concentrations of FGM, generated a resistant HIV-1 strain (IIIBFGMres. virus), with two unique mutations in gp120, namely I153L in the V2 loop and K457I in the C5 region. These two mutations are in the neighbourhood of regions in gp120 involved in the cellular CD4 receptor binding. In addition, our HIV-1 IIIBFGMres. virus showed cross-resistance with the two well-known compounds dextran sulfate and cyclotriazadisulfonamide (CADA), which, respectively, interfere directly and indirectly with the CD4/gp120 interaction (Chapter 7).Likewise, we investigated the antiviral activity of the labyrinthopeptins, that belong to a novel class of lantibiotics (lanthionine-containing antibiotics) (Chapter 8). The most well-known, and so far commercially used, lantibiotic is nisin in the food industry. As far as we know LabyA1 and LabyA2 are the only two described labyrinthopeptins and preliminary data reported a moderate anti-herpes activity in vitro. Based in these results, we investigated these peptides for their anti-HIV activity, as there is more and more evidence that HIV-1 and HSV-2 are important co-pathogens that synergistically cause various diseases. In several cell lines and against various wild type and drug resistant HIV and HSV strains LabyA1 showed a consistent broad-spectrum antiviral activity. LabyA2 demonstrated no anti-HIV activity and a weak anti-HSV activity. Nisin, used as reference lantibiotic, was not able to inhibit any of the evaluated viruses. As described for GRFT and FGM, LabyA1 inhibits also the DC-SIGN mediated route of transmission and fusion between persistently HIV-infected T cells and non-infected CD4+ target T cells. Virus binding and replication experiments demonstrated that LabyA1 interfered with the entry process of HIV-1 and HSV-2. Additional studies showed an interaction of LabyA1 with the surface glycoprotein gp120 and blocked in this way viral entry in a post-CD4 binding event. As HIV-1 and HSV-2 are two co-pathogens, increasing each others infection, LabyA1 could have potential as a microbicidal agent applied for example in a vaginal gel. Therefore, it is necessary to evaluate the safety of LabyA1 on epithelial cells, as in previous clinical trials an increase in HIV transmission was noted due to vaginal epithelial cell toxicity of potential microbicidal candidates (e.g. nonoxynol-9). A microbicide should not harm the epithelial integrity, causing neither inflammation nor stimulation of the viral target cells. When PBMCs were pretreated with LabyA1, in contrast to the mitogenic lectin phytohemagglutinin (PHA), no increase in the expression of the cellular activation markers CD69 and CD25 was seen, nor increased HIV, nor significant induction of various inflammatory cytokines and chemokines. The vaginal bacterial flora forms a natural barrier against various pathogenic intruders by creating an acidic environment. As natural pH-values in the vagina can increase the risk for HIV transmission, it is therefore very important that an effective potential microbicide causes no harm to the vaginal flora. As LabyA1 is an antibiotic peptide, we investigated its effect on the growth of various vaginal Lactobacilli strains and compared it with nisin. These data clearly demonstrate that LabyA1 caused no toxicity in these vaginal Lactobacilli at concentrations up to 120 µM, while nisin killed most of the these Lactobacilli at relatively low concentrations (at 3 µM). This also clearly indicates the discrepancy between the antiviral and antibacterial activity of different members of the class of lantibiotics. Due to its dual antiviral activity, which is highly consistent against both HIV and HSV (in contrast to tenofovir, showing a strongly reduced antiviral activity against HSV-2, compared to HIV-1). LabyA1 can be seen as a potential microbicidal candidate. As microbicides may comprise at least two active agents, we investigated the degree of synergy with other widely clinically used anti-HIV (e.g. tenofovir, raltegravir, saquinavir) and anti-HSV (e.g. acyclovir) drugs. These studies demonstrated no antagonistic drug combinations, increasing again its potential for further microbicidal development.These data clearly show the need to search for an effective microbicide. The class of CBAs could be combined with other antiviral agents which are clinically-approved or under development. The labyrinthopeptins, especially LabyA1, can be seen as a lead peptide for the development of novel and even more potent derivatives with anti-HIV and anti-HSV activity; while novel derivatives of FGM could lead to a new class of small peptidic CD4/gp120 inhibitors.
Inour research, we showed that tenofovir can be combined with various members of the class of CBAs (e.g. HHA, GNA, MVN, BanLec,...) and that this synergy increases their combined antiviral potency and decreases potential cytotoxicity of these agents (Chapter 3).Despite the fact that the glycans present on gp120 are an attractive target for antiviral therapy, there is a huge diversity between the HIV subtypes as such. We specifically investigated the subtypes B and C, as they account for >60% of the total HIV-1 infections worldwide. In a follow-up study, we investigated if griffithsin (GRFT), the most potent anti-HIV-1 CBA described to date, could be combined with other classes of antiretroviral drugs (e.g. integrase inhibitors, entry inhibitors and reverse transcriptase inhibitors) and if there is a complete loss of antiviral activity when certain sugars differ between the included subtypes. It was reported that the N-linked glycans N295 and N234 on gp120 were involved in the interaction with GRFT and we focused mainly on these two glycans in our experiments. We observed that GRFT lost only minor activity when one or even both sugars were absent. Moreover, GRFT showed synergy with the HIV-1 inhibitors maraviroc (CCR5 antagonist), enfuvirtide (gp41 fusion peptide inhibitor), raltegravir (integrase inhibitor) and tenofovir (reverse transcriptase inhibitor) (Chapter 4). Each CBA binds to specific sugar moieties (e.g. mannose or N-aceylglucosamine) and their bridges (e.g. alpha(1,6) or alpha(1,2) linkages). Based on the above mentioned results, we investigated if CBAs as such could be combined against wild type HIV-1 and HIV-2 strains, and also against certain HIV-1 CBAresistant strains (Chapter 5). We performed most of the experiments with GRFT. First, we could prove that GRFT and the banana lectin (BanLec) also possess an equally potent anti-HIV-2 activity. Various (pre-)clinical antiretroviral agents have a strong reduced, if any, antiviral activity against HIV-2 (e.g. enfuvirtide, microvirin (MVN)). We could also demonstrate that GRFT, in combination with various CBAs (such as MVN, BanLec or the broad neutralizing anti-carbohydrate-binding mAb 2G12) inhibited HIV replication more efficiently. Surprisingly, when the CBAs HHA and GNA were combined, rather antagonistic to additive effects were observed against wild type HIV viruses and synergy against CBAresistant HIV-1 strains, which deleted 1 (as seen with HIV-1 NL4.32G12mAbres. virus) to 4 N-linked glycans (in case of HIV-1 NL4.3MVNres. virus). In addition, GRFT and BanLec kept their potent antiviral activity against these two CBAresistant viruses. These results clearly indicate that (i) CBAs could be combined as such, (ii) that they exhibit different binding patterns on the glycans of gp120 and that (iii) affinity and kinetics are crucial factors in their antiviral activity.Semen and cervicovaginal secretions of HIV-infected persons contain cell-free HIV particles and HIV-infected immune cells (e.g. T cells and macrophages). These cells can easily transmit HIV through T cell - T cell contacts to uninfected CD4+ uninfected T cells. The dendritic cells (DCs) are a population of cells, which detect and capture HIV particles upon infection. These cells express DC-SIGN (dendritic cell-specific intercellular adhesion molecule-3 grabbing non-integrin), an attachment receptor for various pathogens such as HIV. Upon receptor interaction the virus is efficiently transmitted to the non-infected CD4+ T cells in the lymph nodes. So, we investigated in more detail if GRFT could also inhibit, alone and in combination with other classes of antiretroviral drugs (namely entry inhibitors, reverse transcriptase inhibitors, integrase inhibitors and protease inhibitors), the HIV-1 transmission/replication and the CD4+ target T cell destruction as well, through these T cell - T cell and DCs - T cell contacts (Chapter 6). These intercellular contacts result in the formation of multinucleated giant cells or syncytia. We were able to visualize how these giant cells were formed in function of time by the JuLI(TM) live cell-imaging viewer and how the CD4+ target T cell population was destroyed. These data also clearly show that GRFT, alone and in combination, prevents both cell-to-cell HIV-1 transmission and destruction of the CD4+ T cell population.In the second part of this thesis, we focused more on the novel antibiotic peptides feglymycin (FGM) and labyrinthopeptins (LabyA1). We investigated more in depth their antiviral spectrum and mechanism of action. FGM, a peptide of 13 amino acids, showed a consistent broad-spectrum anti-HIV activity in inhibiting infection with cell-free HIV particles as well as in cocultivation assays between persistently HIV-infected T cells and CD4+ non-infected T cells. In the DC-SIGN dependent transmission route FGM inhibits not the capture of the virus on DC-SIGN, but subsequently the viral replication upon cocultivation with the CD4+ target T cells. These data clearly indicate that the glycans present on gp120 are not involved in the anti-HIV mechanism of action of FGM. By the use of various virus binding assays (e.g. surface plasmon resonance, ELISAs, flow cytometry) and viral replication studies, we were able to prove that FGM inhibited the CD4/gp120 binding, through interaction with the viral envelope protein gp120. Alanine scanning mutagenesis studies of FGM, where each amino acid is systematically replaced by a neutral alanine, resulted in a significant loss of antiviral activity and affinity for gp120, when the L-aspartate at position 13 was replaced by a neutral alanine. Cultivating HIV-1 in the presence of increasing concentrations of FGM, generated a resistant HIV-1 strain (IIIBFGMres. virus), with two unique mutations in gp120, namely I153L in the V2 loop and K457I in the C5 region. These two mutations are in the neighbourhood of regions in gp120 involved in the cellular CD4 receptor binding. In addition, our HIV-1 IIIBFGMres. virus showed cross-resistance with the two well-known compounds dextran sulfate and cyclotriazadisulfonamide (CADA), which, respectively, interfere directly and indirectly with the CD4/gp120 interaction (Chapter 7).Likewise, we investigated the antiviral activity of the labyrinthopeptins, that belong to a novel class of lantibiotics (lanthionine-containing antibiotics) (Chapter 8). The most well-known, and so far commercially used, lantibiotic is nisin in the food industry. As far as we know LabyA1 and LabyA2 are the only two described labyrinthopeptins and preliminary data reported a moderate anti-herpes activity in vitro. Based in these results, we investigated these peptides for their anti-HIV activity, as there is more and more evidence that HIV-1 and HSV-2 are important co-pathogens that synergistically cause various diseases. In several cell lines and against various wild type and drug resistant HIV and HSV strains LabyA1 showed a consistent broad-spectrum antiviral activity. LabyA2 demonstrated no anti-HIV activity and a weak anti-HSV activity. Nisin, used as reference lantibiotic, was not able to inhibit any of the evaluated viruses. As described for GRFT and FGM, LabyA1 inhibits also the DC-SIGN mediated route of transmission and fusion between persistently HIV-infected T cells and non-infected CD4+ target T cells. Virus binding and replication experiments demonstrated that LabyA1 interfered with the entry process of HIV-1 and HSV-2. Additional studies showed an interaction of LabyA1 with the surface glycoprotein gp120 and blocked in this way viral entry in a post-CD4 binding event. As HIV-1 and HSV-2 are two co-pathogens, increasing each others infection, LabyA1 could have potential as a microbicidal agent applied for example in a vaginal gel. Therefore, it is necessary to evaluate the safety of LabyA1 on epithelial cells, as in previous clinical trials an increase in HIV transmission was noted due to vaginal epithelial cell toxicity of potential microbicidal candidates (e.g. nonoxynol-9). A microbicide should not harm the epithelial integrity, causing neither inflammation nor stimulation of the viral target cells. When PBMCs were pretreated with LabyA1, in contrast to the mitogenic lectin phytohemagglutinin (PHA), no increase in the expression of the cellular activation markers CD69 and CD25 was seen, nor increased HIV, nor significant induction of various inflammatory cytokines and chemokines. The vaginal bacterial flora forms a natural barrier against various pathogenic intruders by creating an acidic environment. As natural pH-values in the vagina can increase the risk for HIV transmission, it is therefore very important that an effective potential microbicide causes no harm to the vaginal flora. As LabyA1 is an antibiotic peptide, we investigated its effect on the growth of various vaginal Lactobacilli strains and compared it with nisin. These data clearly demonstrate that LabyA1 caused no toxicity in these vaginal Lactobacilli at concentrations up to 120 µM, while nisin killed most of the these Lactobacilli at relatively low concentrations (at 3 µM). This also clearly indicates the discrepancy between the antiviral and antibacterial activity of different members of the class of lantibiotics. Due to its dual antiviral activity, which is highly consistent against both HIV and HSV (in contrast to tenofovir, showing a strongly reduced antiviral activity against HSV-2, compared to HIV-1). LabyA1 can be seen as a potential microbicidal candidate. As microbicides may comprise at least two active agents, we investigated the degree of synergy with other widely clinically used anti-HIV (e.g. tenofovir, raltegravir, saquinavir) and anti-HSV (e.g. acyclovir) drugs. These studies demonstrated no antagonistic drug combinations, increasing again its potential for further microbicidal development.These data clearly show the need to search for an effective microbicide. The class of CBAs could be combined with other antiviral agents which are clinically-approved or under development. The labyrinthopeptins, especially LabyA1, can be seen as a lead peptide for the development of novel and even more potent derivatives with anti-HIV and anti-HSV activity; while novel derivatives of FGM could lead to a new class of small peptidic CD4/gp120 inhibitors.
Publication year:2013
Accessibility:Closed