Anhydride-modified Ovalbumin As A Potential Microbicide For Preventing Sexual Transmission Of HIV

Objection:Despite extraordinary advances in the development of prevention and therapeutic strategies against human immunodeficiency virus (HIV) infection, HIV/AIDS continues to spread at an alarming rate worldwide. There are approximately 7,400 new infections and over 5,500 new deaths resulting from AIDS each day. There are three main transmission routes for HIV, including by blood, mother to child and unprotected sex. Unprotected sex is the primary route for humans now, especially for females in many developing countries, to acquire HIV/AIDS. Therefore, development of female-controlled topical microbicides is urgently needed.Micribicides are gels, creams, foams, or suppositories, which can be applied in vagina or rectum before sex to prevent sexual transmission of HIV. These products are designed to protecting HIV infection by killing HIV, inhibiting HIV entry target cells or HIV replication in the cells.An ideal microbicide should be broad antiviral activity, safe, highly effective, affordable, stable in human fluids, and easy to use. It should be safe to human tissues and to vigina bacteria. Microbicides should also be cheap to be used in many developing countries. Based on these characters, our group is searching for some new anti-HIV microbicide candidates for preventing HIV sexually transmission.In 1995, our group of New York Blood Center demonstrated that bovine B-lactoglobulin, a protein present in milk and whey, modified by 3-hydroxyphthalic anhydride (3HP-B-LG) displayed broad antiviral activities against infection by human and simian immunodeficiency viruses (HIV-1, HIV-2 and SIV), and other viral pathogens causing sexually transmitted diseases (STD), such as herpes simplex viruses (HSV-1 and HSV-2). Nether non-modified B-lactoglobulin nor 3-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 of 3HP-B-LG as a microbicide.Therefore, in the present study, we sought to replace bovine proteins with chemically modified animal proteins of non-bovine origin as new anti-HIV microbicide candidates. All of the non-bovine animal proteins including chicken ovalbumin (OVA), rabbit serum albumin (RSA), porcine serum albumin (PSA), gelatin from cold water fish skin (G-FS), gelatin from porcine skin (G-PS) were modified by 3-hydroxyphthalic anhydride (HP), using the same method and the same conditions as 3HP-B-LG. Methods and Results:After extensive screening, we found that several HP-modified non-bovine-origin proteins exhibited inhibitory activity (HP-OVA, HP-RSA, HP-PSA, HP-G-FS, HP-G-PS) against infection by HIV-1 X4 (HIV-1ⅢB) and R5 (HIV-1BaL) viruses. By analyzing the structure of the proteins found to possess antiviral activity, OVA, RSA and PSA were found to have representative globulins identical to bovineβ-lactoglobulin. By contrast, the gelatins used in this study are derived from collagens, which had different structure and conformation. The absence of anti-HIV activities of these modified proteins indicated that HIV blocking abilities might not be solely dependent on the modified lysine or arginine but also on the protein conformation. Thus, the presence of specific globular structures might play an important role in the anti-HIV activity of OVA, RSA and PSA. y evaluating the anti-HIV activities of these modifications and the characteristics of proteins used in the reaction, we found that HP-modified chicken ovalbumin (HP-OVA) was the most promising anti-HIV inhibitor among these modified proteins. Chicken ovalbumin (OVA) is the main protein in egg white, making up 60-65% of the total protein. Since OVA is one of the most abundant proteins consumed by people worldwide and is a generally recognized as a safe (GRAS) protein, HP-modified OVA has great potential for further development as an effective, safe and affordable microbicide.To search for alternate anhydrides as chemical modifiers of OVA, we selected two other anhydrides, succinic anhydride (SU) and maleic anhydride (ML), for the chemical modification. We compared the efficiency of three different anhydrides, including ML, SU, as well as HP, for the chemical modification of OVA. All three anhydrides (SU, ML and HP) were sufficiently potent to convert OVA into an effective anti-HIV agent. By compared the anti-HIV-1 activity of those three anhydride OVAs, HP-OVA and ML-OVA demonstrated more efficacy than SU-OVA in blocking HIV-1 infection, especially the sexually transmitted R5 virus. It is the chemical structure of anhydrates that accounts for the effect of different anhydride OVA modifications on HIV inhibitory activities. Specifically, the only difference between maleic and succinic anhydride was the double bond between C3 and C4 in maleic anhydride, which led to the stronger inhibition abilities of ML-OVA over those of SU-OVA on HIV infection.3-hydroxyphthalic anhydride has a hydrophobic aromatic group, leading to the most potent anti-HIV activity. These findings suggest that the aromatic and unsaturated structure in anhydrides might contribute to the difference in antiviral activities of these modified OVAs.For confirming the optimal condition, we prepared a series of HP-OVA by changing the different HP concentration and different pH values. Here we found that the percentage of the HP-modified and unmodified lysine and arginine residues in OVA was dependent on the concentration of HP and pH of the reaction system and was correlated with the anti-HIV-1 activity of HP-OVA. These results suggested that the modified amino acid residues play an important role in mediating the antiviral activity. We selected 40 mM concentration of HP and pH 8.5 as the optimal condition for preparation of the HP-OVA for the subsequent studies. Under such condition, HP-OVA had 99.86% and 89.26% of the lysine and arginine residues modified by HP, respectively.By ELISA, HP-OVA effectively inhibited infection by laboratory-adapted HIV-1 strains, including X4 and R5 viruses, with IC50 in the nM range. HP-OVA is highly effective in inhibiting the infection of the primary R5 viruses with distinct genotypes and phenotypes, with IC50 from 0.011μM to 0.578μM. HP-OVA is more effective against the predominant HIV-1 subtypes A, B, and C. Particularly, HP-OVA is highly potent against the primary HIV-1 isolated in Thailand,92TH009 (subtype A/E, R5) with IC50 about 10 nM, while it was reported that the HIV-1 subtype A/E R5 virus was preferentially sexually transmitted. Those results suggest that HP-OVA have a great potency to be used as a microbicide candidate in the world, especially in our country.HP-OVA is also effective in inhibiting HIV-2 infection, suggesting that this microbicide candidate may also be applicable in West Africa where HIV-2 is prominent. Our studies also showed that HP-OVA could potently inhibit infection by SHIV and SIV. Since both SHIV and SIV can be used for infection of rhesus macaques, HP-OVA will be tested in a non-human primate model for evaluation of its in vivo efficacy against SHIV or SIV infection through vaginal challenge.Compared with several negatively charged polymeric microbicide candidates failed in clinical trials, most of these microbicides are more effective against X4 than R5 viruses, possibly because X4 viruses have more positively charged residues in the V3 loop of gp120 than R5 viruses. However, this is not the case for HP-OVA since it is almost equally effective against both X4 and R5 viruses. Next, we try to find the mechanism of HP-OVA for inhibiting the HIV infection.By using time-of-addition, cell-cell fusion, and cell-to-cell transmission assays, we demonstrated that HP-OVA 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, SPR and FCM assay, HP-OVA 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. Furthermore, we demonstrated that HP-OVA could block the formation of the fusion-active gp41 six-helix bundle. These results suggest that HP-OVA inhibit HIV-1 entry/fusion through multiple mechanisms of action by interacting with both gp120 and gp41 as well as CD4 via the negatively charged residues of HP-OVA.The failure of microbicide candidates clinical trials warned us that the safety evaluation of a microbicide candidate should be carried out as early as possible. Here we first assessed the potential cytotoxicity of HP-OVA on three well-characterized cell lines derived from the human vaginal and cervical epithelium and three human T immune cell lines as well as PBMCs, which were used for evaluation of the in vitro anti-HIV-1 activity of HP-OVA. The results showed that HP-OVA had low cytotoxicity to all tested cells. Its selectivity index (SI=CC50/IC50) ranged from 253 to 13,066, indicating that HP-OVA appears to be safe in vitro. More extensive animal studies to evaluate its in vivo toxicity will be carried out in the future. Vaginal lactic acid bacteria are not be affected by HP-OVA, even in the high concentration in 22.4μM. Since HP-OVA can bind to sCD4 while the CD4 molecule on the T lymphocytes plays an important role in T cell activation, one of the concerns is whether HP-OVA affects the function of CD4+ T cells or induces T cell anergy, a status of the lymphocyte that is functionally inactivated following an antigen stimulation. Our studies demonstrated that proliferation of T lymphocytes in human PBMCs stimulated with PHA was not significantly affected by HP-OVA at the concentration as high as 100μM. Further studies showed that HP-OVA had no significant effect on the productions of IFN-y by PBMCs with or without PHA stimulation. Those results suggest that HP-OVA may not have deleterious effects on the function of CD4+ T cells, especially for those circulating in the human body. But we cannot exclude the possibility that long-term use of CD4 blockers topically may suppress the function of CD4+ immune cells located in vaginal mucosa. Therefore, long-term observation of the potential harmful effect of HP-OVA on the mucosal immune system is warranted.The disadvantage of protein/peptide drugs, such as T20, is their short half life resulting from the hydrolysis by proteases like trypsin. Since trypsin is the major protease in human and predominantly cleaves peptide chains at the carboxyl side of the amino acids lysine and arginine in human beings, we tested whether HP-modified proteins are sensitive to trypsin. Notably, treatment of HP-OVA with trypsin did not affect its anti-HIV-1 activity, indicating that the HP-modified lysine and arginine residues became resistant to trypsin. Notably, treatment of HP-OVA with trypsin, which predominantly cleaves peptide chains at the carboxyl side of the amino acids lysine and arginine, did not affect the anti-HIV-1 activity of HP-OVA, similar in SDS- PAGE, indicating that the protein containing the HP-modified lysine and arginine residues becomes resistant to trypsin.An ideal microbicide candidate should be active against HIV-1 infection in the presence of human body fluids, such as seminal fluid or cervicovaginal fluid, because the topical microbicides will be applied intravaginally or intrarectally. In the present study, we tested the effects of seminal fluid and vaginal fluid simulant on anti-HIV activities of HP-OVA. The results indicate that the antiviral activities of HP-OVA are stable in the presence of those fluids, suggesting that HP-OVA should be active being used as a microbicide.From 2000 to 2009, the failed clinical trials of several negatively charged polymeric microbicide candidates, such as Savvy (G31G), Cellulose sulfate (CS), 0.5% PRO 2000 gel and Carrageenan, made the research prospect of microbicide candidates is not optimistic. The main possible reason is that these microbicides are more effective against X4 than R5 viruses, possibly because X4 viruses have more positively charged residues in the V3 loop of gp120 than R5 viruses. The failures of these microbicide candidates have turned the focus on the next-generation microbicide candidates containing highly effective anti-HIV drugs in the present, such as TMC-120, UC781 and Tenofovir. One of the limitations of the anti-HIV drugs available in the market currently is the rapid emergence of HIV resistance. A combination of HP-OVA and other anti-HIV drug-based microbicide candidates with different target sites may have the following advantages:(i) maximizing antiviral efficacy, (ii) minimizing toxic effects and high cost due to combining dose reduction, (iii) fighting against diverse anti-HIV-1 drug mutations or delaying the emergence of HIV-1 resistance by using agents with different mechanisms and (iv) low price of production. Most importantly, HP-OVA is highly soluble and stable for long-term storage.Conclusion:By evaluating the anti-HIV activities and analyzing the mechanism of action, we conclude that HP-OVA 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-OVA has promising potential to be developed as an anti-HIV microbicide for preventing HIV sexual transmission. HP-OVA 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-1 strains 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.