34 research outputs found

    Expression in Escherichia coli of a cloned DNA sequence encoding the pre-S2 region of hepatitis B virus

    Get PDF
    A DNA sequence encoding the entire pre-S2 region (amino acids 120-174; serotype ayw) of human hepatitis B virus envelope protein has been inserted into the lacZ gene of the plasmid pSKS105 yielding a recombinant, pWS3. Lac+ colonies of the Escherichia coli M182 (lacIOPZYA), isolated after transformation with pWS3, produced a pre-S2 peptide-ß-galactosidase fusion protein. This fusion protein, which comprised as much as 3% of the total bacterial protein, was purified to >90% homogeneity by affinity chromatography on p-aminophenyl-ß-D-thiogalactoside-Sepharose. It is immunoprecipitable with rabbit antibodies to a synthetic peptide corresponding to amino acids 120-145 of the pre-S2 region of serotype adw [pre-S(120-145)] or with antibodies to hepatitis B virus. pre-S(120-145) completely blocked the binding of either antibody to the pre-S2 peptide-ß-galactosidase fusion protein. These results indicate that there are antigenic determinants on the fusion protein that are closely related to, if not identical to, determinants on synthetic pre-S(120-145) and on pre-S2 sequences of native hepatitis B virus particles. Thus, bacteria transformed with pWS3 can provide an abundant source of pre-S2-ß-galactosidase fusion protein, which may prove useful either as a diagnostic reagent possessing marker enzyme activity suitable for ELISA tests or as an immunogen with potential to contribute to active prophylaxis of hepatitis B

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

    Get PDF
    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

    Water dispersible microbicidal cellulose acetate phthalate film

    Get PDF
    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°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 ≈50 to ≈65% ethanol (EtOH). The films are ≈100 µ 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

    Real-time single-molecule observation of rolling-circle DNA replication

    Get PDF
    We present a simple technique for visualizing replication of individual DNA molecules in real time. By attaching a rolling-circle substrate to a TIRF microscope-mounted flow chamber, we are able to monitor the progression of single-DNA synthesis events and accurately measure rates and processivities of single T7 and Escherichia coli replisomes as they replicate DNA. This method allows for rapid and precise characterization of the kinetics of DNA synthesis and the effects of replication inhibitors

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

    Get PDF
    BACKGROUND: 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 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

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

    Get PDF
    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 α-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

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

    Get PDF
    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 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. 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 ≈ 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. 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

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

    Get PDF
    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 β-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

    Water dispersible microbicidal cellulose acetate phthalate film

    No full text
    Abstract 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°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 ≈50 to ≈65% ethanol (EtOH). The films are ≈100 µ 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.</p
    corecore