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Here, we report that the S-acyl-2-mercaptobenzamide thioester (SAMT) class of human immunodeficiency virus type 1 (HIV-1) nucleocapsid protein (NCp7) inhibitors was able to prevent transmission of HIV-1 from infected cells, including primary cells. Furthermore, when SAMTs were introduced during an HIV-1 challenge of cervical explant tissue, inhibition of dissemination of infectious virus by cells emigrating from the tissue explants was observed. Preliminary studies using a rhesus macaque vaginal challenge model with mixed R5 and X4 simian-human immunodeficiency virus infection found that five of six monkeys were completely protected, with the remaining animal being partially protected, infected only by the R5 virus. These data suggest that SAMTs may be promising new drug candidates for further development in anti-HIV-1 topical microbicide applications.

Cellulose acetate phthalate (CAP, cellulose acetate 1,2-benzenedicarboxylate) is a common polymeric oral tablet coating. CAP is also a vaginal microbicide candidate that potently inhibits HIV-1 proliferation. This paper describes the development of a precise, stability-indicating gel permeation chromatography (GPC) assay for CAP. During accelerated stability studies monitored by separate reversed-phase high performance liquid chromatography (RP-HPLC) and GPC analyses, an apparent loss of mass balance was observed. This deficit was corrected by recalculating the response factor (RF) for each degraded sample, proportional to the fraction of phthalate remaining bound to the polymeric CAP. The correction factor enabled CAP and the degradation product phthalic acid (PA) to be quantitated by a single GPC analysis. The chromatographic approach taken here could potentially apply to any polymer containing degradable chromophores.

Homo- and heterochiral poly(mandelic acid)s (PMDAs) were synthesized under strongly acidic, mildly acidic, and nonacidic conditions. The water-soluble fractions of these polymers were evaluated with respect to their inhibitory activity against the human immunodeficiency virus (HIV-1). Polymers were prepared via a step-growth mechanism, yielding linear polyesters. The polymers were characterized by CHS elemental microanalysis, X-ray fluorescence (XRF), and FT-IR spectroscopy. Polymers prepared by the three methods have different structures. Both elemental microanalysis and XRF indicated the presence of S in those polymers prepared by treatment with concentrated H2SO4, which were the only ones exhibiting inhibitory and virucidal activity against HIV-1, mediated by their binding to cellular co-receptor binding sites on the virus envelope glycoprotein gp120. Additionally, FT-IR spectroscopy indicated the complete absence of C=O functionality in the H2SO4-prepared PMDA.

Background: Evaluation 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., Methods: The 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 beta-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., Results: The 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 approximately 4 to approximately 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., Conclusion: The 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.

Cellulose acetate 1,2-benzenedicarboxylate (CAP), a pharmaceutical excipient used for enteric film coating of capsules and tablets, was previously shown to have potent inhibitory activity against infection by human immunodeficiency virus type 1 (HIV-1) T cell line-adapted (TCLA) strains. In the present study, we determined the inhibitory activity of CAP against infection by cell-free and cell-associated primary HIV-1 isolates with distinct genotypes and biotypes in cervical explants, peripheral blood mononuclear cells (PBMCs), monocytederived macrophages (MDMs), and CEMx174 5.25M7 cells. CAP blocked infection by cell-free and cell-associated HIV-1 in cervical explants. It inhibited infection by cell-free primary HIV-1 isolates (clades A to G and group O) in PBMCs, MDMs, and CEMx174 5.25M7 cells and blocked transmissions of the cell-associated primary HIV-1 isolates from dendritic cells (DCs) to PBMCs, from MDMs to PBMCs, and from PBMCs to CEMx174 5.25M7 cells. The inhibitory activity of CAP on infection by the cell-free and cell-associated primary HIV-1 isolates is independent of viral subtypes and coreceptor usage. These data suggest that CAP is a good microbicide candidate that can be further developed for preventing sexual transmission of HIV-1.

For approximately 24 years the AIDS pandemic has claimed approximately 30 million lives, causing approximately 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.

Objectives: To assess the safety and distribution of a cellulose acetate 1,2-benzenedicarboxylate (CAP) gel formulation in rhesus macaques as part of the development process for its use as a vaginally administered product in humans., Design: The similarities between the reproductive physiology, anatomy and vaginal microflora of human and non-human primates makes non-human primates a relevant animal model to assess the safety and distribution of candidate anti-HIV microbicides., Methods: CAP gel was instilled once or once daily for 4 days into the vaginal vault of rhesus macaques. Colposcopy and magnetic resonance imaging were performed to detect adverse effects and spread of CAP, respectively. Additionally, vaginal pH and composition of the vaginal micorflora in macaques before, during and after CAP instillations were determined, and vaginal biopsies obtained following repeated CAP exposures were examined to further document its safety., Results: CAP is safe for repeated use and exhibits a favorable distribution profile, showing no evidence of penetration into cells that line the vaginal epithelium. Further, the presence of CAP has no adverse effect on vaginal pH or the composition of the vaginal microflora, and does not induce vaginal epithelial thinning or inflammation., Conclusions: CAP gel shows minimal toxicity in vivo, supporting its use as a candidate vaginal microbicide in humans.

Objectives: To evaluate the protective efficacy of cellulose acetate 1,2-benzenedicarboxylate (CAP) formulated in a glycerol-based gel against infection with CXCR4 (X4) and CCR5 (R5) viruses in the simian/human immunodeficiency virus (SHIV)/rhesus macaque model of HIV-1 transmission., Design: Mucosal infection of non-human primates is a reasonable model for use in the investigation of HIV-1 intervention strategies., Methods: Rhesus macaques treated with Depo-Provera 5 weeks prior to challenge were inoculated intravaginally twice, over a period of 6 h with mixed inocula of pathogenic X4- and R5-SHIV in the presence or absence of CAP. Plasma viral load, peripheral and mucosal CD4 T cell counts as well as the genotype of the circulating virus were determined., Results: CAP protected seven of ten macaques against transmission of both X4- and R5-SHIV, reaching statistically significant values (P = 0.0256). Delayed and/or reduced virus replication, as well as blunting of peripheral and mucosal CD4 T cell loss was noted in the three macaques that were infected in the CAP treated group compared to those in the placebo group. Further, protection conferred by CAP appeared to be more effective against X4- than R5-SHIV infection., Conclusions: CAP is protective against highly permissive challenges with X4 and R5 viruses in vivo. Research on further development of this promising compound as a candidate microbicide for the prevention of sexual HIV-1 transmission is therefore warranted.

The combination of two candidate microbicides, cellulose acetate 1,2-benzenedicarboxylate (CAP), a polymer that blocks human immunodeficiency virus type 1 (HIV-1) entry by targeting gp120 and gp41, and UC781, a tight-binding HIV-1 reverse transcriptase inhibitor (RTI), resulted in effective synergy for inhibition of MT-2 cell infection by HIV-1(IIIB), a laboratory-adapted virus strain. The 95% effective concentration values for the combination were reduced about 15- to 20-fold compared with those corresponding to the single compounds. The combination of CAP and UC781 is also synergistic in inhibiting infection of peripheral blood mononuclear cells by a primary HIV-1 isolate, 92US657. Combinations of CAP with other RTIs, such as efavirenz or zidovudine, also had significant synergistic effects on the inhibition of HIV-1 infection. In addition, CAP and UC781 had complementary effects against HIV-1 infection since (i) CAP inhibited infection by the UC781-resistant strain HIV-1(IIIB) A17 and (ii) pretreatment of MT-2 cells with UC781, but not CAP, abolished subsequent infection after removal of the compound. This suggests that the combination of CAP and UC781 represents a promising candidate microbicide for prevention of sexual transmission of HIV-1.

The sexual transmission of human immunodeficiency virus type 1 (HIV-1) is facilitated by inflammation and related epithelial barrier perturbation. Microbicides for vaginal applications are currently being developed to reduce the risk of HIV-1 transmission. However, little is known about their interference with epithelial immune function. In recent clinical trials, nonoxynol-9 (N-9), a virucide with a long history of intravaginal use as a contraceptive, failed to protect against HIV-1 possibly due to mucosal inflammatory damage. Cellulose acetate 1,2-benzenedicarboxylate, also named CAP (for "controls AIDS pandemic"), is an anti-HIV-1 microbicide selected from pharmaceutical excipients that are regarded as safe for oral administration but have not been assessed for potential effects on inflammatory factors in the vaginal environment. Here we use a sensitive human cell culture system to evaluate proinflammatory profiles of soluble CAP in reference to N-9 and known epithelial activators such as tumor necrosis factor alpha (TNF-alpha) and bacterial lysates. Within 6 h of exposure, TNF-alpha and N-9 triggered NF-kappaB and AP-1/cFos activation and upregulated interleukins and an array of chemokines by vaginal and polarized cervical epithelial cells. The induced proinflammatory status continued after removal of stimuli and was confirmed by enhanced transepithelial neutrophil migration. While sustaining stability and anti-HIV-1 activity in the epithelial environment, CAP did not increase the production of proinflammatory mediators during or after exposure, nor did it modify the epithelial resistance to leukocyte traffic. CAP attenuated some TNF-alpha-induced responses but did not interfere with epithelial cytokine responsiveness to gonococcal determinants. The described system may be useful for predicting proinflammatory side effects of other microbicide candidates for vaginal application.

Background: For approximately 24 years the AIDS pandemic has claimed approximately 30 million lives, causing approximately 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., Methods: 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., Results: 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., Conclusion: 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.

Background: Cellulose acetate phthalate (CAP) has been used for several decades in the pharmaceutical industry for enteric film coating of oral tablets and capsules. Micronized CAP, available commercially as "Aquateric" and containing additional ingredients required for micronization, used for tablet coating from water dispersions, was shown to adsorb and inactivate the human immunodeficiency virus (HIV-1), herpesviruses (HSV) and other sexually transmitted disease (STD) pathogens. Earlier studies indicate that a gel formulation of micronized CAP has a potential as a topical microbicide for prevention of STDs including the acquired immunodeficiency syndrome (AIDS). The objective of endeavors described here was to develop a water dispersible CAP film amenable to inexpensive industrial mass production., Methods: CAP and hydroxypropyl cellulose (HPC) were dissolved in different organic solvent mixtures, poured into dishes, and the solvents evaporated. Graded quantities of a resulting selected film were mixed for 5 min at 37 degrees C with HIV-1, HSV and other STD pathogens, respectively. Residual infectivity of the treated viruses and bacteria was determined., Results: The prerequisites for producing CAP films which are soft, flexible and dispersible in water, resulting in smooth gels, are combining CAP with HPC (other cellulose derivatives are unsuitable), and casting from organic solvent mixtures containing approximately equal to 50 to approximately equal to 65% ethanol (EtOH). The films are approximately equal to 100 micron thick and have a textured surface with alternating protrusions and depressions revealed by scanning electron microscopy. The films, before complete conversion into a gel, rapidly inactivated HIV-1 and HSV and reduced the infectivity of non-viral STD pathogens >1,000-fold., Conclusions: Soft pliable CAP-HPC composite films can be generated by casting from organic solvent mixtures containing EtOH. The films rapidly reduce the infectivity of several STD pathogens, including HIV-1. They are converted into gels and thus do not have to be removed following application and use. In addition to their potential as topical microbicides, the films have promise for mucosal delivery of pharmaceuticals other than CAP.

Background: Cellulose 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., Methods: Enzyme 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 beta-gal gene and expressing CD4 and coreceptors CXCR4 or CCR5 were used to measure virus infectivity., Results: 1) 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., Conclusions: Micronized 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.

Background: Cellulose 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., Methods: Enzyme-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 alpha-helical core domain of gp41., Results: 1) 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., Conclusions: CAP 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.

Naturally occurring deletions within the human hepatitis B virus (HBV) preS2 region have frequently been identified in patients with hepatocellular carcinoma (HCC), while chronic carriers without cirrhosis and HCC contain no detectable preS2 deletion variants. We have characterized two different preS2 internal deletion variants from two patients. In addition to several weak phenotypes, our study revealed three unexpected strong phenotypes: (1) a paradoxical "hypermodification" phenomenon was observed with significantly increased size heterogeneity and molecular weights of the secreted middle (M) envelope proteins containing a preS2 internal deletion. This phenomenon was observed in transient transfection with a human hepatoma Huh7 cell line as well as in stable transfection with a rodent hepatoma cell line 7777. (2) A significantly increased intracellular accumulation of all three envelope proteins (large, middle, and small) was detected by both Western blot analysis and immunofluorescence microscopy. (3) The middle envelope proteins with a preS2 internal deletion were not recognized in vitro by a putative neutralizing antiserum, suggesting that these variants can evade immune recognition in vivo. To our knowledge, this is the first identification and characterization of the M deletion variant protein in HBV natural infection.

Background: Cellulose 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., Methods: Enzyme-linked immunoassays and flow cytometry were used to demonstrate CAP binding to HIV-1 and to define the binding site on the virus envelope., Results: 1) 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., Conclusions: These results provide support for consideration of CAP as a topical microbicide of choice for prevention of STDs, including HIV-1 infection.

Initial biologic events that underlie sexual transmission of HIV-1 are poorly understood. To model these events, we exposed human immature Langerhans cells (LCs) within epithelial tissue explants to two primary and two laboratory-adapted HIV-1 isolates. We detected HIV-1(Ba-L) infection in single LCs that spontaneously emigrated from explants by flow cytometry (median of infected LCs = 0.52%, range = 0.08-4.77%). HIV-1-infected LCs downregulated surface CD4 and CD83, whereas MHC class II, CD80, and CD86 were unchanged. For all HIV-1 strains tested, emigrated LCs were critical in establishing high levels of infection (0.1-1 microg HIV-1 p24 per milliliter) in cocultured autologous or allogeneic T cells. HIV-1(Ba-L) (an R5 HIV-1 strain) more efficiently infected LC-T cell cocultures when compared with HIV-1(IIIB) (an X4 HIV-1 strain). Interestingly, pretreatment of explants with either aminooxypentane-RANTES (regulated upon activation, normal T cell expressed and secreted) or cellulose acetate phthalate (potential microbicides) blocked HIV-1 infection of LCs and subsequent T cell infection in a dose-dependent manner. In summary, we document HIV-1 infection in single LCs after exposure to virus within epithelial tissue, demonstrate that relatively low numbers of these cells are capable of inducing high levels of infection in cocultured T cells, and provide a useful explant model for testing of agents designed to block sexual transmission of HIV-1.

Human immunodeficiency virus type 1 (HIV-1) infection continues to spread in developing countries, mostly through heterosexual transmission. The development of a safe and cost-effective topical microbicide, effective against a range of STDs including HIV-1, would greatly impact the ongoing epidemic. When formulated in a vehicle, a micronized form of cellulose acetate phthalate (CAP), which is an inactive pharmaceutical excipient, has been shown to inactivate HIV-1, herpes simplex virus types 1 and 2, cytomegalovirus, Neisseria gonorrhoeae, Trichomonas vaginalis, Haemophilus ducreyi, and Chlamydia trachomatis in vitro. Formulated CAP was also shown to be effective against herpes simplex virus type 2 in vivo. Here we show that a formulation of CAP protected four of six rhesus monkeys from vaginal infection with simian immunodeficiency virus. Thus, CAP may be a candidate for use as a topical microbicide for preventing HIV-1 infection in humans.

Preferred microbicides are expected to inactivate most sexually transmitted viral and nonviral pathogens, including HIV-1, without affecting lactobacilli, components of the natural defense system against sexually transmitted diseases (STDs), be widely available, be inexpensive, and have an established safety record for human use. We show here that cellulose acetate phthalate [C-A-P enteric coating polymer (Eastman)], a compound used for coating of enteric tablets, meets all these criteria.

Heterosexual transmission of human immunodeficiency virus type 1 (HIV-1) is the major cause of the ongoing AIDS epidemic. Application of chemical barrier methods is expected to contribute to the worldwide control of this epidemic. Bovine beta-lactoglobulin modified by 3-hydroxyphthalic anhydride (3-hydroxyphthalovyl-beta-lactoglobulin [3HP-beta-LG]) was shown to inhibit HIV-1, HIV-2, simian immunodeficiency virus (SIV), herpes simplex virus type 1 and 2, and Chlamydia trachomatis infection in vitro. Here, we show that 3HP-beta-LG not formulated into any vehicle protected three of six rhesus monkeys against vaginal infection by SIV. Incorporation of the compound into an appropriate vehicle is expected to increase the degree of protection. 3HP-beta-LG may be effective as a vaginal inhibitor of HIV-1 infection in humans.

Kinetics of 3-hydroxyphthaloyl-beta-lactoglobulin-CD4 interaction were evaluated using a biosensor instrument based on surface plasmon resonance. A very fast association (k(a)=2.4+/-0.3x10(6)M(-1)s(-1)) and slow dissociation (K(d)=2.3+/-0.14x10(-4)s(-1)) rate constants were observed indicating the high affinity of the complex. This result together with earlier data, suggest that "structure-specific" requirements must be met to endow acid anhydride modified lactoglobulin with the capacity for high affinity binding to CD4., (Copyright 1999 The International Association for Biologicals.)

The human immunodeficiency virus (HIV-1) pandemic has been driven primarily by the sexual transmission of the virus, and facilitated by prior infections with other sexually transmitted disease (STD) pathogens. Although treatment of these STDs has been proposed as a means to decrease the rate of HIV-1 sexual transmission, preventive measures effective against both HIV-1 and other STD pathogens are expected to have a larger impact. These measures include topically applied mechanical and chemical (i.e. microbicidal) barriers. Microbicides of preference should have a broad specificity against diverse STD pathogens and a well established safety record, considering their repeated use over decades. Here, we report that cellulose acetate phthalate (CAP), an "inactive" pharmaceutical excipient, commonly used in the production of enteric tablets and capsules: (1) has antiviral activity against HIV-1 and several herpesviruses (HSV); and (2) when appropriately formulated, in micronized form, inactivates HIV-1, HSV-1, HSV-2, cytomegalovirus, Neisseria gonorrhoeae, Trichomonas vaginalis, Haemophilus ducreyi and Chlamydia trachomatis but does not affect Lactobacilli, components of the natural vaginal flora contributing to resistance against STDs. Thus, the CAP formulations meet the criteria for preferred microbicides and warrant further evaluation in vivo in humans., (Copyright 1999 The International Association for Biologicals.)

The spread of sexually transmitted infections caused by herpes simplex virus type 2 (HSV-2) has continued unabated despite educational efforts generated in response to the human immunodeficiency virus (HIV) epidemic. Given the absence of effective vaccines, this indicates the need to develop prophylactic measures such as topical antiviral agents. Chemical modification of bovine beta-lactoglobulin (beta-LG), the major protein of whey, by hydroxyphthalic anhydride (3HP) led to the generation of a potent HIV-1 inhibitor designated 3HP-beta-LG. This agent was shown to also have antiviral activity against HSV-2 and HSV-1 in vitro. Recent studies indicate that 3HP-beta-LG binds to HSV-1 virions, which, at least in part, involves the viral glycoprotein gE. Here we show that 3HP-beta-LG inhibits HSV-2 infection in the mouse model of genital HSV-2 infection. Simultaneous exposure to HSV-2 and 3HP-beta-LG caused a significant decrease in the proportion of infected animals (27% virus shedding, 5% lesion development and 0% fatality for 3HP-beta-LG as compared to 80% shedding, 60% lesion development and 53% fatality in mice treated with PBS). The proportion of animals with HSV-2 infection after treatment with beta-LG was similar to that in the PBS-treated group. Pretreatment with 3HP-beta-LG formulated in a gel, which prolongs the presence of the agent in the vagina, also significantly reduced the proportion of HSV-2-infected mice (5% virus shedding, 5% lesion development and 0% fatality for 3HP-beta-LG as compared to 70% shedding, 60% lesion development and 40% fatality in vehicle-treated mice). These differences were significant (P < or = 0.0005, 0.002 and 0.008 for shedding, lesion development and fatality, respectively). Virus titres in the minority of mice that developed infection were similar to those in untreated mice. HSV-2 infection was not inhibited by treatment of an ongoing infection, indicating that 3HP-beta-LG interferes with the initial infection. These data suggest that 3HP-beta-LG may be an efficacious agent for preventing vaginal transmission of genital herpesvirus infections.

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 beta-lactoglobulin (beta-LG), the major protein of whey, by hydroxyphthalic anhydride (3HP) led to the generation of a potent HIV-1 inhibitor (designated 3HP-beta-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-beta-LG, indicating that this compound: (i) inhibited infection by human cytomegalovirus (HCMV), which is known to be sexually 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-beta-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-beta-LG. In agreement with results on inhibition of HIV-1 infection, 3HP-beta-LG appears to be the acid anhydride-modified protein of choice as an antiviral agent against herpesviruses.

Bovine beta-lactoglobulin chemically modified with 3-hydroxyphthalic anhydride (3HP) was recently shown, at nanomolar concentrations, to block the binding site on CD4 for the HIV surface glycoprotein (gp120), potentially inhibiting HIV transmission. Human glycodelin has sequence homology with bovine beta-lactoglobulin and appears as different glycoforms in endometrium (GdA) and seminal plasma (GdS). We studied the anti-HIV effects of chemically modified GdA and GdS on both the infection of MT-2 cells by HIV-1IIIB, and the infection of peripheral blood mononuclear cells by the primary HIV isolate THA/93/051 belonging to subtype E. Whereas the native proteins were inactive when tested at physiologic concentrations, nanomolar concentrations of either 3HP-GdA or 3HP-GdS inhibited the production of HIV nucleocapsid p24, cytopathic effects of HIV-1IIIB, and infection of peripheral blood mononuclear cells by the primary HIV isolate THA/93/051. Moreover, both modified proteins inhibited gp120-CD4 binding, 3HP-GdS being more potent than 3HP-GdA (p = 0.0042). Because GdA and GdS have the same major protein core, the observed difference in gp120-CD4 binding must depend on the specific glycoform. In view of the previously reported contraceptive activity of GdA, the observed anti-HIV activity induced by its chemical modification should be of special interest in the development of antiviral strategies that may also have contraceptive effects.

Sexual transmission is the most frequent (86%) route of adult HIV-1 transmission worldwide. In the absence of a prophylactic anti-HIV vaccine, other methods of preventing infection should be implemented. Virucidal spermicides have been considered for this purpose, but their application is contraindicated by adverse effects. Anti-HIV drugs or virus-neutralizing monoclonal antibodies are expensive, suggesting that their wide use in topical chemoprophylaxis is unlikely. This emphasizes the importance of developing other methods for preventing HIV transmission. The target cells for sexual and mucosal HIV transmission include T lymphocytes, monocytes/macrophages and dendritic cells. Therefore, compounds blocking HIV-CD4 binding are expected to inhibit virus transmission. In exploring the possibility that chemical modification of food proteins might lead to compounds with anti-HIV-1 activity, we found that bovine beta-lactoglobulin (beta-LG) modified by 3-hydroxyphthalic anhydride (3HP-beta-LG) (1) blocked at nanomolar concentrations the binding to CD4 of human (HIV) and simian (SIV) immunodeficiency virus surface glycoproteins and monoclonal antibodies specific for the HIV binding site on CD4 and (2) inhibited infection by HIV-1, including primary virus isolates, by HIV-2 and by SIV. The inexpensive and widely available source (whey) for production of 3HP-beta-LG suggests its potential application (nonparenteral) for diminishing the frequency of HIV transmission.

Several porphyrin derivatives were reported to have anti-HIV-1 activity. Among them, meso-teta(4-carboxyphenyl)porphine (MTCPP) and other carboxyphenyl derivatives were the most potent inhibitors (EC50 <0.7 mu M). MTCPP bound to the HIV-1 envelope glycoprotein gp120 and to full-length V3 loop peptides corresponding to several HIV-1 isolates but not to other peptides from gp120 + gp41. However, it remained possible that MTCPP bound to regions on gp120 which cannot be mimicked by peptides. Further characterization of the binding domain for MTCPP is important for understanding the antiviral activity of porphyrins and for the design of anti-HIV-1 drugs interfering with functions of the virus envelope. Results presented here show that: (i) deletion of the V3 loop from the gp120 sequence resulted in drastically diminished MTCPP binding, suggesting that the V3 loop is the dominant if not the only target site on gp120; (ii) this site was only partially mimicked by full-length V3 loop peptides; (iii) MTCPP binding to the gp120 V3 loop elicited allosteric effects resulting in decreased accessibility of the CD4 receptor binding site; (iv) the binding site for MTCPP lies within the central portion of the V3 loop (KSIHIGPGRAFY for the HIV-1 subtype B consensus sequence) and does not involve directly the GPG apex of the loop. These results may help in designing antiviral compounds with improved activity.

The chemical transformation of synthetic combinatorial libraries to increase the diversity of compounds of medicinal interest was reported recently. Chemical modification of natural products represents a complementary approach to accomplish this aim. Modification of lysines by aromatic acid anhydrides, preferentially by 3-hydroxyphthalic and trimellitic anhydrides and trimellitic anhydride chloride, converted commonly available proteins (human and bovine serum albumin and casein) into potent inhibitors of (i) binding between the HIV-1 gp 120 envelope glycoprotein and the CD4 cell receptor, probably owing to their binding to CD4, and (ii) infection by HIV-1. Modified bovine milk proteins are also potent HIV-1 inhibitors and may have potential for anti-HIV-1 prophylaxis.

A human monoclonal antibody (MAb) (2F5) neutralizing a variety of laboratory strains and clinical isolates of the human immunodeficiency virus type 1 (HIV-1) and binding to an epitope of the envelope glycoprotein gp41 encompassing the amino acid sequence ELDKWA has been described (Muster T et al., J Virol 1993;67:6642-6647). It was suggested that an immunogen eliciting virus-neutralizing antibodies having a specificity similar to that of MAb 2F5 should be considered as a component of HIV-1 vaccines. Efforts in this direction would benefit from understanding the mechanism whereby MAb 2F5 neutralizes the infectivity of HIV-1. The segment of gp41 encompassing residues ELDKWA has so far not been directly implicated in initiation of infection by HIV-1, suggesting that MAb 2F5 might affect other sites on HIV-1 envelope glycoproteins playing a role in virus entry into target cells. We provide here evidence that MAb 2F5 binding to HIV-1 virus particles decreases the accessibility or conformation of the gp41 fusion domain and of gp120 domains, including the binding site for the CD4 cell receptor. These apparently indirect consequences of MAb 2F5 binding to HIV-1 are likely to account for or contribute to the virus-neutralizing activity of this MAb.

Annexin V and apolipoprotein H, reported to bind hepatitis B surface antigen (HBsAg) and presumed to react specifically with the HBsAg S protein and to play an important role in initiation of infection by hepatitis B virus, did not bind to delipidated HBsAg (dlHBsAg). Binding activity was restored by adding lipids to dlHBsAg. These results are consistent with the established affinity of annexin V and apolipoprotein H for lipids.

Using comparative molecular field analysis (CoMFA), a 3D-QSAR model was developed for 21 porphyrin derivatives which have anti-HIV-1 activity and bind to the V3 loop of the envelope glycoprotein gp120 of the human immunodeficiency virus type 1. A significant PLS cross-validated r2cv (0.590) was obtained, indicating that the model could be used as a predictive tool for further design of porphyrin analogs. The model revealed at least three important sites for favorable electrostatic interactions and indicated favorable and unfavorable steric interaction sites. It was found that the occurrence of at least three positively charged and several hydrophobic amino acid residues is highly conserved at fixed positions of gp120 V3 loop sequences. This may support the validity of the proposed model and the hypothesis that porphyrins containing anionic and hydrophobic groups may interact with some of the highly conserved positively charged and hydrophobic sites, respectively, of the V3 loop. These interactions may induce conformational changes in the gp120 envelope glycoprotein leading to inhibition of virus entry into cells and of syncytium formation (cell-to-cell fusion) and thus to inhibition of virus replication.