[Preliminary investigation on the relation between clinical progress and anti-small monomolecular peptides antibody in individual infected with HIV].

OBJECTIVE: To study the quantity of anti-R7V in individuals infected with HIV and AIDS patients and its relation with the progression of disease. METHODS: ELISA and precipitation and other methods were used to investigate the quantity of anti-R7V in asymptomatic long-term survivors and AIDS patients. RESULTS: Positive rate and quantity of anti-R7V were higher in the HIV active ones and AIDS. It showed that the quantity and positive rate of anti-R7V were rather high in dissolving test. CONCLUSIONS: It is strong suggestion for anti-R7V to obstruct the replication of virus by interfering the connection between HIV with CCR5 or CXCR4 and so it impossible HIV entering to CD4+ T cells.

In vitro anti-HIV activity of sulfated cell-wall polysaccharides from gametic, carposporic and tetrasporic stages of the Mediterranean red alga Asparagopsis armata.

The gametic, carposporic and tetrasporic reproductive stages from the Mediterranean red alga Asparagopsis armata contain peculiar sulfated galactans with galactose:3,6-anhydrogalactose:sulfates molar ratio of 1:0.01:1.23, 1:0.04:0.47 and 1:0.01:1.13, respectively. These water-soluble polysaccharides were studied for their in vitro activity against the human immunodeficiency virus (HIV-1). Gametic and tetrasporic galactans inhibit HIV replication at 10 and 8 micrograms/ml, respectively, as measured by HIV-induced syncitium formation as well as reverse transcriptase activity in cell-free culture supernatant. The carposporic polysaccharide is ineffective, even at 100 micrograms/ml. The maximal antiviral effect involves the presence of the polysaccharides after or during infection but not before infection. This time of action suggests an inhibition of an early step of HIV infection.

New 3'-azido-3'-deoxythymidin-5'-yl O-(omega-hydroxyalkyl) carbonate prodrugs: synthesis and anti-HIV evaluation.

Prodrugs of zidovudine (AZT) have been synthesized in an effort to enhance its uptake by HIV-1 infected cells and its anti-HIV activity. The 5'-OH function of AZT was functionalized with various enzymatically labile alkyl groups using specific procedures. The prodrug moieties included 5'-O-carbonate, 5'-O-carbamate, and 5'-O-ester. Analogues of the 3'-azido-3'-deoxythymidin-5'-yl O-(omega-hydroxyalkyl) carbonate series were particularly interesting since they were rearranged through an intramolecular cyclic process during their enzymatic hydrolysis. Evidence of this prodrug rearrangement was confirmed by comparison of the serum half-lives of 5'-O-carbonate prodrugs with their corresponding 5'-O-ester- and 5'-O-carbamate-AZT prodrugs. Interestingly, the anti-HIV-1 activities (EC(50)) of 3'-azido-3'-deoxythymidin-5'-yl O-(4-hydroxybutyl) carbonate 10 in acutely infected MT-4 cells and in peripheral blood mononuclear cells (PBMCs) were 0.5 nM and 0.78 nM, respectively. Compound 10 was 30 to 50 times more potent than its parent drug AZT. Our results suggest that the specific intramolecular rearrangement associated with the 3'-azido-3'-deoxythymidin-5'-yl O-(omega-hydroxyalkyl) carbonate prodrugs could explain the remarkable anti-HIV-1 activity of this series of AZT prodrugs. Prodrug 10 may therefore have better clinical potential than AZT for the treatment of AIDS.

Use of proline bioisosteres in potential HIV protease inhibitors: phenylalanine-2-thiophenoxy-3-pyrrolidinone: synthesis and anti-HIV evaluation.

The synthesis of new phenylalanine-2-thiophenoxy-3-pyrrolidinones is described. Anti-HIV recombinant protease assays and HIV infected cell culture assays (observation of syncytia) demonstrated the potent anti-HIV activity of this new class of pseudopeptides.

Synthesis and anti-HIV evaluation of new 2',3'-dideoxy-3'-thiacytidine prodrugs.

A series of anti-HIV prodrugs possessing various polyaminated side arms have been developed. The incorporation of a N-Boc protected monoamine or diamine side arm into the backbone of the 2',3'-dideoxy-3'-thiacytidine 1 (BCH-189) provided an increase in antiviral potency, which could be several orders magnitude greater than the parent drug (1) depending on the cell culture systems used (MT-4 or MDMs). Twenty six 2',3'-dideoxy-3'-thiacytidine prodrugs which differ from each other by the length, the nature of the 5'-O function and the 5'-O or/and N-4 position on the nucleoside moiety were synthesized. Among this new series of prodrugs, several congeners (12c and 12a) were found to inhibit HIV-1 replication in cell culture with 50% effective concentrations EC50 of 10 and 50 nM respectively, in MT-4 cells. Compound 12c was found more active on infected MDMs cells with 50% effective concentration of 0.01 nM. The synthesis and the antiviral properties of these compounds are discussed.

New anti-HIV derivatives: synthesis and antiviral evaluation.

A small focused library of 18 compounds incorporating the motif 1,3-(N,N'-dibenzyl)diamino-2-propanol has been synthesized, using adapted synthetic methodologies. These series of compounds were evaluated for their in vitro anti-HIV activity on infected MT4 cells (syncytium formation observation). Some of the new synthesized compounds show potent anti-HIV activities. EC50 values for compounds (31, 40, 34, 37 and 46) range from 0.1 to 1 microM. In order to determine at which level these new derivatives interfere with the HIV replicative cycle, inhibition assays on recombinant HIV protease and HIV integrase have been performed. None of the compounds were found active on these two enzymatic targets. Experiments are in progress in order to identify their biological target within the HIV replicative cycle.

[Research of status of provirus HIV DNA in non-progressor and progressor of HIV-1 infected persons].

OBJECTIVE: This study was designed to investigate the different status of provirus HIV DNA in non-progressor and progressor of HIV-1 infected persons. METHODS: HIV RNA, HIV DNA were detected with long-distance PCR (LD-PCR), reverse transcription PCR (RT-PCR), Southern blot and molecular hybridization from plasma and peripheral blood mononuclear cells (PBMCs), using primers locating between long terminal repeats LTR(U5)-LTR(R) and so on. RESULTS: 9.1 kb band is full-length HIV DNA genome which was present in all templates of 12 progressors. Deleted HIV DNA was observed in 9 of 12 progressors. Both the full-length and defective. provirus HIV DNA was detected in 5 of the 18 non-progressors. Defective HIV DNA was only detected in the other non-progressors. CONCLUSIONS: Clinical stages are closely related with both the full-length and the defective HIV DNA.

Antiviral and anticoagulant activities of a water-soluble fraction of the marine diatom Haslea ostrearia.

A water-soluble fraction from the marine diatom Haslea ostrearia was capable to inhibit the in vitro replication of HSV-1 in Vero cells with 50% inhibitory concentration (EC50) of 14 micrograms/ml at a multiplicity of infection of 0.01 ID50/cells. In addition, this fraction delayed the HIV-1-induced syncitia formation on MT4 cells. At concentrations up to 200 micrograms/ml, no cytotoxicity was observed for both the Vero and MT4 cells. The fraction only inhibited the blood coagulation process at concentrations considerably exceeding the EC50.

Synthesis and biological evaluation of polyaminated 2',3'-dideoxy-3'-thiacytidine prodrugs.

The syntheses and biological evaluation of polyaminated 2',3'-dideoxy-3'-thiacytidine have been performed. A new lead was found to increase the in vitro antiviral potency (syncitia formation on MT-4 cell line) of two order magnitude greater than the parent nucleoside drug. Moreover, the in vitro activity on HIV macrophages was found to be more than 3 log greater than the activity of the parent drug 1.

New polyazamacrocycle-nucleoside conjugates: synthesis, anti-HIV evaluation and interaction with CXCR-4 coreceptor.

We report the synthesis of new conjugates that incorporate in their structure bis-tetraazamacrocycle coupled with AZT via enzymolabile bond. Two series of bis-polyazamacrocycles-AZT conjugates were designed, synthesized and evaluated for their antiviral effect in vitro as well as their capability to bind to CXCR-4 coreceptor.

Swainsonine protects both murine and human haematopoietic systems from chemotherapeutic toxicity.

The haematopoietic system is sensitive to cytotoxic damage and is often the site of dose-limiting toxicity. We previously reported that swainsonine, an inhibitor of protein glycosylation, reduced the bone marrow toxicity resulting from a single dose of anticancer drugs in otherwise healthy mice. However, more important questions are (1) can swainsonine protect tumour-bearing mice without interfering with the anti-tumour effects of the drugs, and (2) can swainsonine stimulate haematopoietic activity of human, as well as murine, bone marrow. We demonstrate here that swainsonine protects C57BL/6 mice bearing melanoma-derived tumours from cyclophosphamide-induced toxicity without interfering with the drug's ability to inhibit tumour growth. Similar results were obtained in vivo with 3'-azido-3'-deoxythymidine (AZT), a myelosuppressive agent often used in therapy for acquired immune deficiency syndrome. Swainsonine increased both total bone marrow cellularity and the number of circulating white blood cells in mice treated with doses of AZT that typically lead to severe myelosuppression. Swainsonine also increased the number of erythroid and myeloid colony forming cells (CFCs) in short-term cultures of murine bone marrow, restoring the number of progenitor cells to the control level in the presence of AZT doses that reduced CFCs by 80%. With respect to the sensitivity of human haematopoietic cells to swainsonine, we show that swainsonine protected human myeloid progenitor cells from AZT toxicity in vitro. These results suggest that swainsonine may be useful as an adjuvant in several types of human chemotherapy.

A novel epitope R7V common to all HIV-1 isolates is recognized by neutralizing IgG found in HIV-infected patients and immunized rabbits.

We have previously reported that the presence of antibodies (Ab) directed to the beta2-microglobulin-derived peptide R7V in patient's serum correlated with the nonprogression to AIDS. In order to investigate whether R7V motif could represent a potential target for neutralization, we have immunopurified anti-R7V Ab from sera of nonprogressors, as well as from sera of rabbits injected with R7V. We showed that human as well as rabbit purified,anti-R7V IgG precipitated laboratory adapted strains, as well as primary isolates from different clades indicating that: (1) R7V epitope is a common motif presented at the surface of genetically divergent HIV-1 strains (2) R7V is immunogenic in vivo. When used in neutralizing assay, purified anti-R7V Ab from human or rabbit origin were shown to neutralize infection by HIV-1 laboratory adapted strains and HIV-1 primary isolates. All together, our results indicate that the R7V motif shared by all HIV strains could be considered as a possible candidate for an HIV vaccine.

Rationale for a vaccine using cellular-derived epitope presented by HIV isolates.

It has been clearly demonstrated that cellular antigens (HLA, beta 2-microglobulin) are incorporated at the virion surface. The same epitope derived from beta 2-microglobulin is presented on all virus isolates. The peptide was identified by blocking the neutralizing capacity of a monoclonal antibody directed to R7V epitope: using this peptide for developing an ELISA, we have detected antibodies in nonprogressor patients with neutralizing property to laboratory strains and primary isolates. Purified anti-R7V antibodies immunoprecipitate all HIV isolates at concentration dependent. R7V is immunogenic after rabbit immunization and induces HIV immunoprecipitating and neutralizing antibodies. The patient's as well as the immunized rabbit antibodies did not bind to any cell. No autoimmune disease is found in nonprogressor patients. For all these reasons, R7V is a good candidate for an universal AIDS vaccine.

New bicyclam-AZT conjugates: design, synthesis, anti-HIV evaluation, and their interaction with CXCR-4 coreceptor.

We report the synthesis of mono- and bis-tetraazamacrocycle-AZT conjugates. All new compounds were screened for their ability to inhibit HIV-1 replication in MT4 cell line and were compared to AZT alone. It appears that N-protected covalent prodrugs are equipotent to AZT as inhibitor of HIV replication, while N-deprotected analogues exhibit both higher activity and selectivity against HIV-infected cells. The most active antiviral compounds 27, 28, 34, and 35 were then tested for their binding capability to CXCR-4 receptor. N-Boc analogues 27 and 34 were only weakly effective; in contrast, N-deprotected conjugates 28 and 35 were antagonists to 12G5 mAb binding until 0.05 and 5 microg/mL, respectively. The stability of compound 28 in human plasma was evaluated, and half-life was found to be approximately 8 h in the described conditions. All these results seem to demonstrate the confidence of our prodrug approach, with analogue 28 emerging as the best candidate as lead compound in HIV-1 polytherapy perspective.

Synthesis and anti-HIV activity of alpha-thiophenoxy-hydroxyethylamide derivatives.

A series of new anti-HIV derivatives containing a novel alpha-thiophenoxyhydroxyethylamide core have been synthesized, using S-phenylbenzenethiosulfonate as the thiosulfenylating reagent. Some of the new synthesized compounds (1a, 1c, 1g, 1i, 1j and 1l) inhibited HIV replication in cell culture assays (syncytia formation) with effective concentrations (EC(50)) ranging from 0.1-1 microM. Incorporation of thiophenoxy substitution within various pseudomimetic peptide backbones provided a series of highly potent HIV inhibitors.

Experimental conditions that increase the production of HIV-1 by monocyte-derived macrophages: use of collagen matrix.

Monocyte-derived macrophages (MDMs) from healthy blood donors were isolated by adherence to tissue culture-treated plasticware. They were cultured in vitro in medium supplemented with human serum and recombinant GM-CSF, then infected with the macrophage-tropic prototype strain HIV-1-PAR. Virus production was quantitated at various times after infection by measuring reverse transcriptase concentration in cell-free tissue culture supernatant fluids, using a sensitive nonradioactive assay. Virus production was significantly increased by culturing MDMs on plasticware previously coated with collagen 1. The increase in virus production was dependent upon collagen 1 concentration, with maximal value being encountered after coating with 1.5 microg/cm2. These results indicate that the sensitivity of peripheral macrophages to HIV-1 infection might be influenced by contact-dependent interactions involving components of the extracellular matrix that take place during the process of monocyte extravasation and migration.

Sexual and mother-to-child transmission of the human immunodeficiency virus type 1: a review.

PROBLEM: Sexual and mother-to-child transmission of the human immunodeficiency virus (HIV) type 1 occurs only with a low percentage of infection. Many instances of sexual intercourse result in no transmission, and only 20% of children are infected from seropositive mothers (3% in mothers treated with azidothymidine). METHOD OF STUDY: We analyzed the presence of HIV in various ejaculates of the same HIV-infected patients, as well as in the cervico-vaginal fluid. We have studied the mechanism of transmission from mother to child, by analyzing the cell-to-cell transmission in the trophoblast. RESULTS: Some ejaculates collected at different times from the same HIV-infected males are free of virus, explaining the low rate of sexual transmission. We never found HIV in mobile spermatozoa. The trophoblast can be infected by HIV with a strain dependence and also transiently. By analyzing the tissue of the fetus, it was found that only some organs are infected, confirming the cell-to-cell transmission between the mother and child and not a true vertical transmission through the germinal lines. CONCLUSIONS: HIV is not always present in the genital secretion, explaining the low rate of sexual transmission. Mother-to-child transmission occurs during pregnancy but often after the second trimester and at delivery after cell-to-cell or blood transmission, respectively.

Primary intestinal epithelial cells can be infected with laboratory-adapted strain HIV type 1 NDK but not with clinical primary isolates.

Infectivities of HIV-1 primary isolates and laboratory-adapted strains were compared in primary fetal enterocytes and the colonic epithelial cell line HT29. Infection by two laboratory strains, HIV-1 NDK and HIV-1 NDK(A4), which were adapted on CEM and HT29 cells, respectively, produced significant amounts of virus in both target cell systems. Intestinal cells were resistant to infection with HIV-1 primary isolates regardless of their genetic subtype or SI/NSI phenotype. Biological properties of analyzed viruses rather than differences in cultivation system seem to be responsible for differences between these in vitro and ex vivo results.