Antiviral Activity And Safety Evaluation Of Anhydride-modified Human Serum Albumin

Since the first cases of HIV/AIDS were reported in1981in the United States, AIDS is spreading worldwide rapidly. Theoretically, the best method to preventing HIV infection is to develop an HIV vaccine. However, there is still no effective HIV vaccine to be used in human beings. Currently, the major treatment for preventing HIV infection is the antiviral drugs. Until now,29antiviral drugs with different mechanism of actions have been approved by Food and Drug Administration (FDA). Most of them are belongs to two classes. One is reverse transcriptase inhibitor and the other is protease inhibitor. In spite of so many kinds of anti-HIV drugs used in patients, HIV can not be killed clearly. Those drugs can only suppress viral replication, reduce viral load and partly restore a patient’s immune function and prolong the lives of patients. In2003, the first HIV entry inhibitor, Enfuweite (T-20), was approved by FDA, which is a peptide derived from HIV envelope protein gp41. HIV entry inhibitor can prevent the virus invasion at the first stage. However, it only specifically inhibits HIV-1infection but not inhibiting HIV-2infection. Otherwise, peptide drugs have expensive price, which also limit the clinical application. Another thorny issue is the high mutation of HIV virus. It is extremely easy to produce drug-resistant strains, which can reduce the antiviral activities of most of antiviral drugs. The highly active antiretroviral combination therapy (Highly Active Anti-Retroviral Treatment, HAART) is always combining with three or four kinds of antiviral drugs (typically containing a protease inhibitor). It has become a standard method of treating HIV infection, which is significantly reduced the mortality and morbidity of HIV-infected patients. HAART is also called as "cocktail therapy". A combination of different mechanisms of antiviral drugs may have the following advantages:(i) maximizing antiviral efficacy because of the potent synergistic activity against HIV-1laboratory-adapted both X4and R5strains,(ii) minimizing toxic effects and high cost due to combining dose reduction, and (iii) fighting against diverse anti-HIV-1drug mutations or delaying the emergence of HIV-1resistance by using agents with different mechanisms. Therefore, the devlepment of effective, safe and affordable new antiviral drugs to preventing HIV transmission is urgently needed.Human serum albumin (HSA) is the most common protein in human plasma. It has a lot of important functions of physiology and pharmacology. Now HSA has become one of the most extensive target proteins in basic and clinical research, especially on appropriate chemical modification. Development a chemically modified product as an ideal therapeutic molecule has become one of the important means of research in life sciences. The non-glycosylated single polypeptide chain of HSA contains585amino acids and a relative molecular weight was66.5kD. It is mainly composed of18kinds of amino acid residues, in which contains59lysine and27arginine. Since the anhydride is a strongly acidic substance, the protein containing many alkaline amino acids can be modified by the anhydride. After chemical modification, an anhydride-modfied protein will be produced.In1995, Neurath AR and Shibo Jiang in our group of New York Blood Center demonstrated that bovine β-lactoglobulin, a protein present in milk and whey, modified by3-hydroxyphthalic anhydride (3HP-β-LG) displayed broad antiviral activities against infection by human and simian immunodeficiency viruses (HIV-1, HIV-2and SIV), and other viral pathogens causing sexually transmitted diseases (STD), such as herpes simplex viruses (HSV-1and HSV-2). Nether non-modified B-lactoglobulin nor3-hydroxyphthalic anhydride had the anti-HIV activity. It means the chemical modification converted commonly available proteins into potent antiviral compounds.3HP-β-LG is highly stable in aqueous solution for long-term storage at room temperature and elevated temperatures. However, the outbreak of bovine spongiform encephalopathy (BSE) in Europe raised a safety concern with regard to developing bovine proteins for medical use, resulting in discontinuation of further development of3HP-β-LG as a microbicide.HS A is the main protein in human blood, and it is composed of more than60%of the total proteins in the plasma. The structure of HSA is stable and it widely distributed in the human vascular and extravascular circulation. The half life of HSA is up to19days. Because of its human-derived protein, it is no obvious immunogenicity. Therefore, the anhydride modified HSA has the superiority to be developed an ideal antiviral drug in future. Therefore, we sought to replace bovine proteins with chemically modified HSA using the same method and the same conditions as3HP-β-LG Its antiviral activity and its possible clinical applications will be evaluated in the present study.Optimization of the conditions for chemical modification of HP-HSAHP-HSA was prepared according to the procedures previously described. For Optimization of the conditions for chemical modification of HP-HSA, we prepared HP-HSA under six different sets of conditions and evaluated the correlation between the anti-HIV-1activities (both X4and R5) and the percentage of lysine and arginine residues in HP-modified and-unmodified HSA to optimize the conditions for production of HP-HSA. Results showed that, with the increased concentration of HP (20,40and60mM, pH9.0) and the pH of the reaction system (7.0,8.0and9.0, concentration of HP is60mM), a higher percentage of lysine and arginine residues was modified, leading, in turn, to stronger anti-HIV-1activity on both IIIB (X4strain) and BaL (R5strain) viruses. Based on these results, we selected as the optimal condition (60mM of HP at pH9.0) for preparation of the HP-HSA. Under such condition, the EC50s of HP-HSA are about0.851and1.454nM on ⅢIB and BaL viruses, with98.17%and91.67%of the lysine and arginine residues modified by HP, respectively (Table1).Determination of the antiviral activities of HP-HSA It is desirable that a topical antiviral drug should be a broad-spectrum and effective antiviral agent. By ELISA, HP-HSA effectively inhibited infection by laboratory-adapted HIV-1strains, including X4and R5viruses, with IC50in the nM range.HP-HSA is also effective in inhibiting HIV-2infection, suggesting that this microbicide candidate may also be applicable in West Africa where HIV-2is prominent. Our studies also showed that HP-HSA could potently inhibit infection by SIV. Results showed that HP-HSA strongly inhibited the infection by HIV-2ROD, and SIVmac25132H viruses, with low EC50s in the range of8.582to65.769nM Since SIV can be used for infection of rhesus macaques, HP-HSA will be tested in a non-human primate model for evaluation of its in vivo efficacy against SIV infection through vaginal challenge..Still, one of the most prominent problems facing future antiviral drugs is the development of resistant HIV-1strains against reverse transcriptase inhibitors. Our evidence demonstrated that HP-HSA retained high activities against different resistant HIV-1variants, including NRTI-resistant Zidovudine-R, non-nucleoside reverse transcriptase inhibitor (NNRTI)-resistant A17-R, and HIV-1entry inhibitor-resistant NL4-3(36G)V38A and NL4-3(36G)V38E/N42S (Table1). Those results indicate that HP-HSA is capable of preventing the sexual transmission of HIV-1strains that are resistant to the currently used antiretroviral therapeutics.As we described above, herpes simplex virus-2(HSV-2) produces most genital herpes, which is primarily associated with HIV transmission. Clinical data indicate that patients with prevalent HSV-2infection have a two to four-fold higher risk of HIV acquisition than HSV-2uninfected population. Therefore, we investigated the efficacy of HP-HSA against HSV-2in vitro in this study. Results showed that HP-HSA also has very low inhibitory activity on HSV-2and the ECsos value was34.514nM. The negative control protein, unmodified HSA even at concentrations up to10.000uM, had no inhibitory activity against any of the above viruses (data not shown). Cell viability analysis results showed that HP-HSA at the concentration of the effective concentration for100%inhibition (EC100) and lower than EC100had no cytotoxicity to virus target cells (Vero cells).Evaluation the possible mechanism of HP-HSA on HIV-1infection.As described before, HP-HSA exhibited significantly decreased inhibitory activity when it was added to target cells0.5-2h post-HIV-1infection by time of addition assay.6While such results indicated that HP-HSA was an HIV-1entry/fusion inhibitor, the potential targets of HP-HSA remained unknown. Therefore, to systematically search for these targets, we examined the effect of HP-HSA on the early steps of the HIV-1replication cycle, i.e., HIV-1fusion/entry, using cell-cell fusion and virus-cell fusion assays.HIV-1Env--mediated cell-cell fusion was detected using fusion of noninfectious CHO-WT cells with MT-2cells or infectious HIV-1ⅢB-infected H9cells (H9/HIV-1Ⅲb cells) with MT-2cells, respectively. In noninfectious analysis system, the giant syncytia were observed by an inverted microscope. The size of syncytium is usually twice or more times than the size of normal cells. In infectious analysis system, the fluorescence diffuses were observed by an inverted fluorescence microscope. The numbers of the formation of giant syncytia and fluorescence diffuses in cell-cell fusion assay were counted by choosing four fields randomly under an inverted microscope. HP-HSA could significantly decrease the formation of giant syncytia and fluorescence diffuses in a dose-dependent manner. The percentage of inhibition of cell-cell fusion of HP-HSA and HSA were calculated according to the formula described above. Results showed that HP-HSA inhibited both kinds of HIV-1Env-mediated cell-cell fusion, with EC50about27.82nM in a noninfectious system and7.45nM in an infectious system, respectively. In the above-described assays, unmodified HSA demonstrated no ability to inhibit cell-cell fusion.A single-round entry assay was chosen to detect the inhibition of HP-HSA on direct virus-cell fusion by using pseudotyped viruses expressing HIV-1HXB2(X4strain) and SF162(R5strain) Env. Similar results were observed in this assay. HP-HSA inhibited infection by both X4and R5Env-pseudotyped viruses, with EC50about1.51and1.54nM, respectively. Vesicular stomatitis virus-G (VSV-G) pseudovirus was produced by cotransfecting VSV envelope G-protein plasmid and pNL4-3E-R-Luc plasmid. Therefore, VSV-G pseudovirus could be used to evaluate the specific effects of HP-HSA on HIV-1envelope proteins. Results showed that VSV-G pseudovirus infection could not be inhibited by HP-HSA, even at the concentration as high as1.25Ｌ.MCell-to-cell transmission is the major route for the spread of HIV-1. Here, we detected the inhibition of HP-HSA on the transmission of cell-associated HIV-1virions from PBMCs to CEMxl745.25M7cells. HP-HSA blocked the cell-cell HIV-lBaL transmission between those two kinds of virus target cells, which suggests that it could prevent the transmission of cell-associated HIV-BaL isolates. The EC50of cell-cell transmission was31.01nM. Unmodified HSA had no inhibitory activity on cell-cell transmission.All of these results indicate that HP-HSA can inhibit HIV-1infection by blocking the virus entry step. HIV-1entry into a CD4+T cell is mediated by viral envelopes, which comprise the surface subunit gpl20and transmembrane subunit gp41. Similar to HP-OVA and ML-OVA, one of the probable mechanisms of HP-HSA might be interference with the viral envelope glycoprotein or the receptor on the target cell membrane.Evaluation the safty of HP-HSAgeneral, HIV transmission during sexual intercourse should be occurred in presence of normal human body fluids, such as seminal and vaginal secretions. The antiviral activities of an ideal antiviral drug should be retained in the presence of those human body fluids. Here, we determined the potential effect of SP and VFS on the anti-HIV activity of HP-HSA. Results showed that neither SP nor VFS had significant effect on the inhibitory activity of HP-HSA against infection by both HIV-1X4(ⅢB) and R5(BaL) strains. The EC50values of HP-HSA on HIV-1ⅢB strains in the presence of PBS, SP or VFS were1.605nM,1.532nM or1.408nM, respectively. Moreover, the EC50values of HP-HSA for inhibiting HIV-1BaL infection in the presence of SP and VFS were4.997nM and7.564nM, respectively, whereas that of PBS control is6.477nM. Lactobacilli are the predominant organisms in the healthy vagina of women, which maintain a normal vaginal microbiota and form a natural physical barrier against other bacteria or viruses. Here, we chose17strains of normal human vaginal lactobacilli and evaluated the effects of HP-HSA on the proliferation of those vaginal lactobacilli. Results showed that HP-HSA had not produced significant inhibitory effect on17strains of vaginal lactobacilli (MIO15.000μM). The MICs of positive control Ampicillin on different strains were from0.841μM to6.730μM.Normal CD4+T lymphocytes in human beings are essential in the immune system, such as helping antigen presenting cell expresses an antigen on MHC class II through a combination of cell-to-cell interactions or through cytokines.32As we described above, since HP-HSA can bind to cellular CD4receptor, a concern is raised about the function of CD4+T lymphocytes in the presence of HP-HSA.33In our previous studies, we confirmed that HP-HSA had no significant effects on the proliferation of T lymphocytes and no obvious effects on the production of IFN-y in either human PBMCs or PHA-stimulated PBMCs. One advantage of HP-HSA is its origin from human sera, which may reduce any deleterious effects on the function of CD4+T cells, especially for those circulating in the blood stream. Nonetheless, the long-term use of CD4blockers could still suppress the function of CD4+T cells located in vaginal mucosa. Therefore, long-term observation of the potential harmful effect of HP-HSA on the mucosal immune system is warranted.Conclusion:By evaluating the anti-HIV activities and analyzing the mechanism of action, we conclude that HP-HSA is broad-spectrum HIV entry/fusion inhibitors through blocking viral entry. By its broad antiviral potency, resistance to trypsin hydrolysis, easy preparation, low production costs, wide availability and absence of carcinogenic phthalic group, HP-HSA has promising potential to be developed as an anti-HIV microbicide for preventing HIV sexual transmission. HP-HSA may be used in combination with an NNRTI-based microbicide for preventing sexual transmission of HIV because the combination may have synergistic antiviral activity against a broad spectrum of HIV-1strains and reduce the potential toxic effects.These findings might be helpful to looking for a novel strategy for treatment of HIV/AIDS and might provide a rational basis for testing of microbicide combinations in vivo.