The Influence Factors And Inhibitors Of The Formation Of SEVI Amyloid Fiber

Currently, heterosexual transmission is the major way that people acquire HIV. The development of microbicides is one of the few biologically sound prevention strategies with the potentialgreatly empower women to protect themselves from HIV. But so far, there has been no effective product in the market yet. Until now, seven phase Ⅱ/Ⅲ clinical trials of microbicide candidates have been completed. The results of clinical trials have been disappointing. What makes these microbicide candidates failure in the clinical trials?Microbicides are products applied inside the vagina, therefore, it could not ignore the effect of semen on microbicides. In 2007, Munch and colleagues discovered that a degradation product of human semen prostatic acidic phosphatase (PAP), corresponding to residues 248-286 in the PAP sequence, is termed semen-derived enhancer of viral infection (SEVI). SEVI can greatly increase human immunodeficiency virus (HIV) infection. The study confirmed that SEVI promotes HIV virus infection. The possible mechanism is that SEVI fibril has strong cationic properties, through two ways to increase the HIV infection: 1) SEVI enhances viral infection by serving as a polycationic bridge that neutralizes the negative charge repulsion that exists between HIV virions and target cells.2) SEVI directly binds HIV through electrostatic contacts and pull down HIV particles, increases the interaction between virus and host cells significantly, which resulting in the facilitate virion attachment and fusion with target cells. Subsequent studies have identified two additional types of amyloid fibrils that are present in semen and enhance HIV infection. The first type is formed by a peptide derived from an N-proximal fragment of PAP (PAP85-120). The second type is derived from physiologically cleaved coagulum proteins in semen which termed semenogelins (SEMs). Our previous study speculated that SEVI amyloid fibrls may the main factor of the failure of polyanions in clinical trials.Most known seminal amyloid fibrils enhance HIV infection and originate from the host environment. However, the formation of amyloid fibrils requires very stringent conditions, such as the vigorous agitation of the monomeric peptides derived from PAP or SEMs for several hours at 37℃.In our previous study, we found that the peptide derived from the co-receptor binding site of HIV-1 MN envelope can antagonist the anti-HIV activity of T-20. To clarify the T20 may has the mechanism of multi target anti HIV fusion, we speculated that T-20 might target both the gp120 co-receptor region and gp41 transmembrane region to inhibit viral enter. However, our in-depth research failed in making any evidence to support the hypothesis. Our recent study showed thesederived peptides, such as polypeptide EP2, could assemble spontaneously into stable amyloid fibrils, without harsh conditions as SEVI and the SEMI (86-107) formed amyloid fibrils. This kind of derived peptides can significantly promote the formation of SEVI and SEM1(86-107) amyloid fibrils. A research in 2007 showedthat native HIV-1 virus envelope protein gp 120 exposed in rats hepatocytes, resulting in released gp120 fragments(INMWQG) into the incubation medium.Coincidentally, the fragments (INMWQG) and HIV-1 gp120 derived peptide EP2 has highly repetitive sequence. We speculated that INMWQG is likely to promote SEVI amyloid fibrils formation and enhance HIV-1 infection. EP2 fragments whether real existence in vivo is not known in the current study, but the short peptide (INMWQG) has been proved to be true in the presence of gp 120 degradation fragments of rats in vivo. In order to development ideal microbicide candidates for preventing HIV sexually transmission, we study the mechanism of SEVI or SEM1(86-107) promoting virus infection. We asked whether HIV gp 120 derived peptides servering as a kind of nucleus (seed), which promote the formation of seminal amyloid fibrils.And is this also one of the reasons for the the failure of microbicide in clinical trials?The discovery of SEVI not only can be used as one of important evaluation index of the future microbicide candidate, but also provides a new target for the development of drugs for prevention and treatment of AIDS. Theoretically, a compound blocks the binding of HIV and amyloid fibrils or prevents the formation of amyloid fibrils, which can reduce the enhancement of viral infection by those fibrils. Anti SEVI antibody can specific binding with SEVI through its antibody domain, thereby antagonizing SEVI enhanced virus infection. Ourgroup confirmed this hypothesis, but anti SEVI antibody shows no antiviral activity. It had been demonstrated that several proteins modified by anhydride showed broad antiviral activities against HIV-1 infection.We previously reported that 3-hydroxyphthalic anhydride (HP) modified chicken ovalbumin (OVA) and human serum albumin (HSA) exhibited broad and highly effective anti-HIV activities as microbicide candidates for anti sexual transmission of HIV. Those studies showed that an inactive protein might be changed to an effective antiviral compound dramatically by anhydride modification on the site-specific amino acids. If we modified API with suitable anhydrides, the anhydride modified API might be developed to a potential and bifunctional microbicide candidate with both anti-HIV activities and anti-SEVI enhancing activities. We also have preliminary preparation a rabbit anti HIV-1 gp41 polyclonal antibody, the antibody significantly improve the antibody antiviral activity after anhydride modified, and can keep its antibody partly functionally active region. Therefore, when anti SEVI polyclonal antibody was anhydride modified, it may keep the antibody activity domain to antagonize SEVI enhancing virus infection effect, thus has a dual function microbicide candidate for high anti HIV activity. It maximizing avoid the possibility failure of future clinical trials due to the existence of SEVI in semen. The chemical modification of anti SEVI polyclonal antibody is expected to solve the present problems and development difficulties of anionic polymer type microbicide, and become a kind of ideal microbicide.In the present study, the project is divided into two parts:Part 1 Peptides derived from the HIV-1 gp120 coreceptor-binding region promotes formation of PAP248-286 amyloid fibrils to potentially enhance HIV-1 infectionUsing biophysical and biochemical methods, we furtherstudy the interaction of theHIV-1 derived peptides with seminal peptides.We analysis themechanismof EP2 promoting the formation of SEVI and SEM1(86-107) fibrils thusenhancing virus infection. We study whether the short peptide (INMWQG) could enhance HIV infection and promote SEVI, SEM1(86-107) amyloid fibrils formation. The study will provide a more reasonable explanation for the formation of amyloid fibrils thus promoting HIV infection, andit also provide a theoretical basis for development of clinical microbicides and anti HIV drug research.The size and the zeta potential of the EP2 peptide diluted into water were determined using a Zetasizer Nano ZS instrument. The self-assemble ability of EP2 was evaluted using a ThT fluorescence assay, TEM. Using TEM,ThT fluorescence assay, Congo red staining assay and circular dichroism (CD) spectroscopy, we found thatEP2 greatly accelerates PAP248-286 amyloid fibril formation. PAP248-286-formed amyloid fibrils with the assistance of EP2 retained the morphology of SEVI. PAP248-286-formed amyloid fibrils with assistance of EP2 enhanced binding of HIV-1 virions to target cells by flow cytometry and fluorescence microscopy. Using HIV-1 viruses infection assay and a single-cycle HIV-1 infection assay, we confirmed that PAP248-286-formed amyloid fibrils with the assistance of EP2 retained the ability to enhance HIV-1 infection.Using similar methods describe above:the size and the zeta potential of the SEMA peptide diluted into water were determined using a Zetasizer Nano ZS instrument. The self-assemble ability of SEMA was evaluted using a ThT fluorescence assay,Congo red staining assay and TEM. SEMA could enhance HIV-1 infection by virus infection assay and fluorescence microscopy. SEMA binding of HIV-1 virions to target cells or SEMA fibrils directly bind to HIV-1 particles by fluorescence microscopy.Then, we examine whether SEMA accelerated amyloid fibrils formation by PAP248-286 or SEMI (86-107). Using Tht fluorescence method and Congo red staining assay,we found that SEMA significantly promoted amyloid fibrils formation by PAP248-286 or SEMI (86-107) peptide.Using virus infection experiments, we confirmed that amyloid fibrils with the assistance of SEMA retained the ability to enhance HIV-1 infection.The HIV-1 gp120 degradation products, such as EP2, in semen may act as viral factors to promote HIV-1 sexual transmission mediated by semen amyloid fibrils or SEVI.1. EP2, a 15-residue short peptide derived from the HIV-1 gp120 coreceptor-binding region could self-assemble into nanofibers, and accelerate the formation of amyloid fibrils by PAP fragments, thus enhacing HIV infection.2. A native HIV-1 virus envelope protein gp120 exposed in rats hepatocytes, resulting in released gp120 fragments(INMWQG) into the incubation medium. INMWQG could enhance HIV-1 infection, promote SEVI or SEMI (86-107) amyloid fibrils formation and mediated the enhancing of HIV-1 infection.Semen-derived Enhancer of Virus Infection (SEVI) can enhance HIV infection. Our previous studies showed that SEVI may be one of the important factors resulting in failure of clinical trials of the polyanionic microbicides for preventing sexual transmission of HIV. Therefore, SEVI should be one of the key targets in developing preventive anti-HIV microbicides. Recently, we demonstrated that rabbit anti-SEVI IgG (API) could effectively reduce SEVI-mediated enhancing effect of HIV infection. But API itself had no significant anti-HIV activity. We previously found that anhydride-modified ovalbumin (OVA) and many other proteins exhibited strong anti-HIV activity. Based on these findings, we design and prepare an anhydride-modified anti-SEVI antibody as a dual-functional microbicide for preventing HIV sexual transmission. It is expected that the anhydride-modified anti-SEVI antibody can not only strongly bind to HIV through its increased negative charge and inhibit HIV replication, but also effectively reduce SEVI-mediated HIV enhancing effect via its anti-SEVI domains. We will evaluate the anti-HIV activity of anhydride modified anti-SEVI antibody in the presence of semen or SEVI peptide. Furthermore, we will study the interaction between anhydride modified anti-SEVI antibody and SEVI, and analyze its effect on SEVI amyloid fibrils formation. This study will provide a foundation for developing anti-HIV prevention drugs.We have demonstrated that both API and HP-API can specifically bind to PAP248-286 and SEVI amyloid fibril in a concentration-dependent manner by ELISA and Western Blot assay. The results suggested that API and HP-API should have the ability that antagonism SEVI amyloid fibril enhancement effect of HIV virus. It is noteworthy that the anhydride modification reduced the binding of API to PAP248-286 and SEVI amyloid fibril, but still retains part of the binding function. Moreover, after anhydride modification, HP-API showed extremely high inhibitory activities on laboratory-adapted HIV-1 strains and primary HIV-1 isolates with EC50 values in low μg/ml range. It is noteworthy that HP-API exhibited similar antiviral activities against the wild-type HIV-1 stains and the drug-resistant HIV-1 strains.By using time-of-addition, cell-cell fusion, and cell-to-cell transmission assays, we demonstrated that HP-API is an HIV entry/fusion inhibitor since it exhibited significantly decreased inhibitory activity when it was added after HIV-1 infection and it showed potent inhibitory activity on cell-to-cell fusion mediated by X4 virus and cell-to-cell transmission of R5 virus. Using ELISA and FCM assay, HP-APIcould block the binding of the HIV-1 Env surface subunit gp120 (from both X4 and R5 viruses) or an anti-CD4 antibody to sCD4, the primary receptor for HIV, resulting in inhibition of interaction between gp120 and CD4. These results suggest that HP-API inhibit HIV-1 entry/fusion through multiple mechanisms of action by interacting with gp120, gp41 and CD4 via the negatively charged residues of HP-API.We also investigated the safety of HP-API. Due to most of the microbicides local effect in vagina, so we detected the cytotoxicities of HP-API on the target cells and the vaginal epithelial cells (VK2/E6E7) by MTT assay. Results showed that HP-API is safety with low toxic, which makes it possible to apply into clinical trial. The results showed that HP-API had low cytotoxicity to all tested cells.HP-API or API shows high-affinity binding to the PAP248-826 or SEVI. This binding could not be interrupted by negative charge of the anionic polymer such as heparin. HP-API or API inhibited PAP248-286 aggregation in a concentration-dependent fashion by Tht and Congo red assay. In particular, high inhibitor levels completely abrogated the formation of fibrils. To more directly monitor the action of HP-API or API on the formation of SEVI, we next performed analyses at the ultrastructural level by using an ultrathin sectioning technique in combination with transmission electron microscopy. HP-API or API (50 μg/ml) completely abrogated the formation of SEVI. By using circular dichroism (CD) assay, we further confirmed that HP-API or API indeed abrogated the formation of SEVI. We next also confirmed that fibers of PAP248-286 generated in the presence of the HP-API or API able to promote HIV-1 infection. Taken together, the results demonstrated that the HP-API or API targets PAP248-286-derived amyloid fibrils. We further found that HP-API and API could not decomposited mature SEVI by Thioflavin T (ThT) methods. HP-API and API shown inhibiting effect in the aggregation process of amyloid fibrils but not degradation of mature fibrils. Using fluorescence confocal microscopy, virus pull-down assay, we found that SEVI or semen promoted the aggregation of HIV-1 particles, and this aggregation effect can be inhibited by HP-API.By evaluating the anti-HIV activities and the mechanism of HP-API, we found that HP-API is broad-spectrum HIV entry/fusion inhibitor through blocking viral entry and binding with SEVI amyloid fibrils. HP-API can inhibit SEVI-mediated enhancement of HIV-1 infection by blocking the binding of SEVI and HIV-1 virions competitively, which make it deserve to be developed a potential and bifunctional microbicide candidate for preventing HIV sexual transmission.1. HP-API is an ideal microbicide candidate for preventing HIV sexual transmission:it is easy preparation, low cytotoxicity and low production cost. HP-API exhibited highly inhibitory activity against infection by laboratory-adapted and primary HIV-1 strains. HP-API showed potent inhibitory activity against infection by drug-resistant HIV-1 strains.2. HP-API could block the binding of the HIV-1 Env surface subunit gp120 (from both X4 and R5 viruses) or an anti-CD4 antibody to sCD4, the primary receptor for HIV, resulting in inhibition of interaction between gp120 and CD4. HP-API or API shows high-affinity binding to the PAP248-826 or SEVI. This binding could not be interrupted by negative charge of the anionic polymer such as Heparin. HP-API or API inhibited PAP248-286 aggregation in a concentration-dependent fashion, then inhibit SEVI-mediated enhancement of viral infection. HP-API could not depolymerize SEVI amyloid fibrils. HP-API inhibited the binding of SEVI fibrils or semen and HIV virions then inhibit SEVI-mediated enhancement of viral infection.