Yun-Yao Li

Punica granatum (pomegranate) juice provides an HIV-1 entry inhibitor and candidate topical microbicide.

For ∼24 years the AIDS pandemic has claimed ∼30 million lives, causing ∼14,000 new HIV‐1 infections daily worldwide in 2003. About 80% of infections occur by heterosexual transmission. In the absence of vaccines, topical microbicides, expected to block virus transmission, offer hope for controlling the pandemic. Antiretroviral chemotherapeutics have decreased AIDS mortality in industrialized countries, but only minimally in developing countries. To prevent an analogous dichotomy, microbicides should be acceptable, accessible, affordable, and accelerative in transition from development to marketing. Already marketed pharmaceutical excipients (inactive materials of drug dosage forms) or foods, with established safety records and adequate anti‐HIV‐1 activity, may provide this option. Therefore, fruit juices were screened for inhibitory activity against HIV‐1 IIIB using CD4 and CXCR4 as cell receptors. The best juice was tested for inhibition of: (1) infection by HIV‐1 BaL, utilizing CCR5 as the cellular coreceptor, and (2) binding of gp120 IIIB and gp120 BaL, respectively, to CXCR4 and CCR5. To remove most colored juice components, the adsorption of the effective ingredient(s) to dispersible excipients and other foods was investigated. A selected complex was assayed for inhibition of infection by primary HIV‐1 isolates. The results indicate that HIV‐1 entry inhibitors from pomegranate juice adsorb onto corn starch. The resulting complex blocks virus binding to CD4 and CXCR4/CCR5 and inhibits infection by primary virus clades A to G and group O. Therefore, these results suggest the possibility of producing an anti‐HIV‐1 microbicide from inexpensive, widely available sources, whose safety has been established throughout centuries, provided that its quality is adequately standardized and monitored.

Anti-HIV-1 activity of anionic polymers: a comparative study of candidate microbicides

BackgroundCellulose acetate phthalate (CAP) in soluble form blocks coreceptor binding sites on the virus envelope glycoprotein gp120 and elicits gp41 six-helix bundle formation, processes involved in virus inactivation. CAP is not soluble at pH < 5.5, normal for microbicide target sites. Therefore, the interaction between insoluble micronized CAP and HIV-1 was studied. Carbomer 974P/BufferGel; carrageenan; cellulose sulfate; dextran/dextrin sulfate, poly(napthalene sulfonate) and poly(styrene-4-sulfonate) are also being considered as anti-HIV-1 microbicides, and their antiviral properties were compared with those of CAP.MethodsEnzyme linked immunosorbent assays (ELISA) were used to (1) study HIV-1 IIIB and BaL binding to micronized CAP; (2) detect virus disintegration; and (3) measure gp41 six-helix bundle formation. Cells containing integrated HIV-1 LTR linked to the β-gal gene and expressing CD4 and coreceptors CXCR4 or CCR5 were used to measure virus infectivity.Results1) HIV-1 IIIB and BaL, respectively, effectively bound to micronized CAP. 2) The interaction between HIV-1 and micronized CAP led to: (a) gp41 six-helix bundle formation; (b) virus disintegration and shedding of envelope glycoproteins; and (c) rapid loss of infectivity. Polymers other than CAP, except Carbomer 974P, elicited gp41 six-helix bundle formation in HIV-1 IIIB but only poly(napthalene sulfonate), in addition to CAP, had this effect on HIV-1 BaL. These polymers differed with respect to their virucidal activities, the differences being more pronounced for HIV-1 BaL.ConclusionsMicronized CAP is the only candidate topical microbicide with the capacity to remove rapidly by adsorption from physiological fluids HIV-1 of both the X4 and R5 biotypes and is likely to prevent virus contact with target cells. The interaction between micronized CAP and HIV-1 leads to rapid virus inactivation. Among other anionic polymers, cellulose sulfate, BufferGel and aryl sulfonates appear most effective in this respect.

Cellulose acetate phthalate, a common pharmaceutical excipient, inactivates HIV-1 and blocks the coreceptor binding site on the virus envelope glycoprotein gp120

BackgroundCellulose acetate phthalate (CAP), a pharmaceutical excipient used for enteric film coating of capsules and tablets, was shown to inhibit infection by the human immunodeficiency virus type 1 (HIV-1) and several herpesviruses. CAP formulations inactivated HIV-1, herpesvirus types 1 (HSV-1) and 2 (HSV-2) and the major nonviral sexually transmitted disease (STD) pathogens and were effective in animal models for vaginal infection by HSV-2 and simian immunodeficiency virus.MethodsEnzyme-linked immunoassays and flow cytometry were used to demonstrate CAP binding to HIV-1 and to define the binding site on the virus envelope.Results1) CAP binds to HIV-1 virus particles and to the envelope glycoprotein gp120; 2) this leads to blockade of the gp120 V3 loop and other gp120 sites resulting in diminished reactivity with HIV-1 coreceptors CXCR4 and CCR5; 3) CAP binding to HIV-1 virions impairs their infectivity; 4) these findings apply to both HIV-1 IIIB, an X4 virus, and HIV-1 BaL, an R5 virus.ConclusionsThese results provide support for consideration of CAP as a topical microbicide of choice for prevention of STDs, including HIV-1 infection.

Role of seminal plasma in the anti-HIV-1 activity of candidate microbicides

BackgroundEvaluation of microbicides for prevention of HIV-1 infection in macaque models for vaginal infection has indicated that the concentrations of active compounds needed for protection by far exceed levels sufficient for complete inhibition of infection in vitro. These experiments were done in the absence of seminal plasma (SP), a vehicle for sexual transmission of the virus. To gain insight into the possible effect of SP on the performance of selected microbicides, their anti-HIV-1 activity in the presence, and absence of SP, was determined.MethodsThe inhibitory activity of compounds against the X4 virus, HIV-1 IIIB, and the R5 virus, HIV-1 BaL was determined using TZM-bl indicator cells and quantitated by measuring β-galactosidase induced by infection. The virucidal properties of cellulose acetate 1,2-benzene-dicarboxylate (CAP), the only microbicide provided in water insoluble, micronized form, in the presence of SP was measured.ResultsThe HIV-1 inhibitory activity of the polymeric microbicides, poly(naphthalene sulfonate), cellulose sulfate, carrageenan, CAP (in soluble form) and polystyrene sulfonate, respectively, was considerably (range ≈ 4 to ≈ 73-fold) diminished in the presence of SP (33.3%). Formulations of micronized CAP, providing an acidic buffering system even in the presence of an SP volume excess, effectively inactivated HIV-1 infectivity.ConclusionThe data presented here suggest that the in vivo efficacy of polymeric microbicides, acting as HIV-1 entry inhibitors, might become at least partly compromised by the inevitable presence of SP. These possible disadvantages could be overcome by combining the respective polymers with acidic pH buffering systems (built-in for formulations of micronized CAP) or with other anti-HIV-1 compounds, the activity of which is not affected by SP, e.g. reverse transcriptase and zinc finger inhibitors.

Anti-HIV-1 activity of cellulose acetate phthalate: Synergy with soluble CD4 and induction of "dead-end" gp41 six-helix bundles

BackgroundCellulose acetate phthalate (CAP), a promising candidate microbicide for prevention of sexual transmission of the human immunodeficiency virus type 1 (HIV-1) and other sexually transmitted disease (STD) pathogens, was shown to inactivate HIV-1 and to block the coreceptor binding site on the virus envelope glycoprotein gp120. It did not interfere with virus binding to CD4. Since CD4 is the primary cellular receptor for HIV-1, it was of interest to study CAP binding to HIV-1 complexes with soluble CD4 (sCD4) and its consequences, including changes in the conformation of the envelope glycoprotein gp41 within virus particles.MethodsEnzyme-linked immunosorbent assays (ELISA) were used to study CAP binding to HIV-1-sCD4 complexes and to detect gp41 six-helix bundles accessible on virus particles using antibodies specific for the α-helical core domain of gp41.Results1) Pretreatment of HIV-1 with sCD4 augments subsequent binding of CAP; 2) there is synergism between CAP and sCD4 for inhibition of HIV-1 infection; 3) treatment of HIV-1 with CAP induced the formation of gp41 six-helix bundles.ConclusionsCAP and sCD4 bind to distinct sites on HIV-1 IIIB and BaL virions and their simultaneous binding has profound effects on virus structure and infectivity. The formation of gp41 six-helical bundles, induced by CAP, is known to render the virus incompetent for fusion with target cells thus preventing infection.

Punica granatum (Pomegranate) juice provides an HIV-1 entry inhibitor and candidate topical microbicide

BackgroundFor ≈ 24 years the AIDS pandemic has claimed ≈ 30 million lives, causing ≈ 14,000 new HIV-1 infections daily worldwide in 2003. About 80% of infections occur by heterosexual transmission. In the absence of vaccines, topical microbicides, expected to block virus transmission, offer hope for controlling the pandemic. Antiretroviral chemotherapeutics have decreased AIDS mortality in industrialized countries, but only minimally in developing countries. To prevent an analogous dichotomy, microbicides should be: acceptable; accessible; affordable; and accelerative in transition from development to marketing. Already marketed pharmaceutical excipients or foods, with established safety records and adequate anti-HIV-1 activity, may provide this option.MethodsFruit juices were screened for inhibitory activity against HIV-1 IIIB using CD4 and CXCR4 as cell receptors. The best juice was tested for inhibition of: (1) infection by HIV-1 BaL, utilizing CCR5 as the cellular coreceptor; and (2) binding of gp120 IIIB and gp120 BaL, respectively, to CXCR4 and CCR5. To remove most colored juice components, the adsorption of the effective ingredient(s) to dispersible excipients and other foods was investigated. A selected complex was assayed for inhibition of infection by primary HIV-1 isolates.ResultsHIV-1 entry inhibitors from pomegranate juice adsorb onto corn starch. The resulting complex blocks virus binding to CD4 and CXCR4/CCR5 and inhibits infection by primary virus clades A to G and group O.ConclusionThese results suggest the possibility of producing an anti-HIV-1 microbicide from inexpensive, widely available sources, whose safety has been established throughout centuries, provided that its quality is adequately standardized and monitored.

3-Hydroxyphthaloyl-β-Lactoglobulin. I. Optimization of Production and Comparison with other Compounds Considered for Chemoprophylaxis of Mucosally Transmitted Human Immunodeficiency Virus Type 1

Modification of the major bovine whey protein, β-lactoglobulin (β-LG) by 3-hydroxyphthalic anhydride (3HP) leads to the generation of a potent inhibitor of infection by human immunodeficiency virus (HIV) types 1 and 2, designated 3HP-β-LG. 3HP-β-LG also has antiviral activity against herpesviruses, albeit at concentrations exceeding those required for inhibition of HIV-1 infection. The topical application of 3HP-β-LG to decrease the rate of sexual transmission of HIV and other sexually transmitted viruses worldwide is being considered. Results presented here: (i) define the conditions for chemical modification of β-LG by 3HP, resulting in 3HP-β-LG with optimum anti-HIV-1 activity; (ii) show that β-LG, prior to chemical modification, or 3HP-β-LG can be exposed to the elevated temperatures used to pasteurize milk without adversely affecting anti-HIV-1 activity; (iii) provide evidence that 3HP-β-LG is a more potent anti-HIV-1 compound than sulphated polysaccharides, other candidate compounds considered as prophylactic agents to prevent sexual transmission of HIV-1; and (iv) confirm that the primary target for 3HP-β-LG is CD4, although binding to the HIV-1 envelope protein gp120 was also observed and contributed to the antiviral activity of 3HP-β-LG.

3-Hydroxyphthaloyl β-lactoglobulin. III. Antiviral activity against herpesviruses †

The spread of sexually transmitted diseases, including human immunodeficiency virus type 1 (HIV-1) and herpesvirus infections, has continued unabated despite educational efforts spearheaded as a response to the HIV-1 epidemic. This suggests the need for prophylactic measures, including the application of topical antiviral agents. Chemical modification of bovine β-lactoglobulin (β-LG), the major protein of whey, by hydroxyphthalic anhydride (3HP) led to the generation of a potent HIV-1 inhibitor (designated 3HP-β-LG) shown to also have activity against herpes simplex virus types 1 and 2 (HSV-1, HSV-2). This report provides more detailed results concerning the anti-herpesvirus activity of 3HP-β-LG, indicating that this compound: (i) inhibited infection by human cytomegalovirus (HCMV), which is known to besexually transmitted; (ii) inactivated the infectivity of both HSV-1 and HSV-2; (iii) inhibited cell-to-cell transmission of HSV-1 and HSV-2; and (iv) bound to HSV-1, HSV-2 and HCMV virus particles and partially inhibited the binding of anti-glycoprotein E (gE) and anti-gC monoclonal antibodies to HSV-1 and HSV-2. The binding of 3HP-β-LG to the herpesviruses under study was inhibited by aggregated human IgG, suggesting that the respective viral Fc receptor is one of the target sites for 3HP-β-LG. In agreement with results on inhibition of HIV-1 infection, 3HP-β-LG appears to be the acid anhydride-modified protein of choice as an antiviral agent against herpesviruses.

3-Hydroxyphthaloyl-β-Lactoglobulin. II. Anti-Human Immunodeficiency Virus Type 1 Activity in in Vitro Environments Relevant to Prevention of Sexual Transmission of the Virus

It is anticipated that the rate of sexual transmission of viruses could be substantially decreased by the use of topical chemical barrier methods. Chemical modification of bovine (β-lactoglobulin (β-LG), the major protein of whey, led to the generation of a potent inhibitor (designated 3HP-β-LG) of human immunodeficiency virus type 1 (HIV-1) infection which was also active against herpesviruses. Compounds intended for topical application to prevent sexual transmission of viruses need to maintain their antiviral activity at pH <<7, corresponding to an acidic vaginal environment, and in the presence of seminal fluid. Results presented here show that the binding of 3HP-β-LG to the CD4 receptor for HIV, involved in the anti-HIV-1 activity of this compound, decreases with decreasing pH. The presence of seminal fluid also decreased the binding of 3HP-β-LG to CD4 and diminished the inhibitory effect of the compound on CD4-gp120 binding. 3HP-β-LG was shown to bind Zn++, and the inhibitory effect of seminal fluid could be substantially diminished by chelating Zn++ with ethylenediaminetetraacetate. Saliva had no effect on 3HP-β-LG binding to CD4 or on its interference with gp120-CD4 binding. The decreased 3HP-β-LG-CD4 binding and the concomitant reduction of gp120-CD4 binding inhibition by 3HP-β-LG at low pH and in the presence of seminal fluid could be compensated for by an increase of the 3HP-β-LG concentration and by adding Zn++ chelators to 3HP-β-LG. These results provide a background for the design of 3HP-β-LG formulations for topical use.