Pertussis toxin B-oligomer dissociates T cell activation and HIV replication in CD4 T cells released from infected lymphoid tissue

OBJECTIVE: To investigate, in human lymphoid tissue infected with HIV-1 ex vivo, the immunostimulatory and HIV inhibitory properties of pertussis toxin B oligomer (PTX-B) and of the genetically modified non-toxic PT-9K/129G. METHODS: Human tonsils from uninfected donors were infected ex vivo with R5 or X4 HIV-1 in the presence or absence of PTX-B. Virus replication was evaluated in culture supernatants; cells emigrated from tissue blocks were immunostained for lymphocytic and activation markers. HIV DNA and cell proliferation were evaluated with real-time PCR and [H]thymidine incorporation, respectively. RESULTS: Both PTX-B and PT-9K/129G inhibited HIV-1 replication. These compounds activated and stimulated the proliferation of emigrated cells, most of which were CD4 T lymphocytes. Cells emigrated from infected tissues did not produce detectable virus in unstimulated or in PTX-B- or PT-9K/129G-stimulated cultures whereas robust virus production was triggered by phytohemagglutinin (PHA) or interleukin-2 (IL-2). Analysis of HIV DNA content indicated that infected cells were present among emigrated cells and that their number greatly increased following IL-2 stimulation, whereas it remained constant in the presence of PTX-B or PT-9K/129G. CONCLUSIONS: PTX-B and PT-9K/129G inhibit both R5 and X4 HIV-1 replication in human lymphoid tissue ex vivo. In contrast to PHA and IL-2, they promote the proliferation of CD4 T lymphocytes emigrated from tissue, including HIV-infected cells, without triggering virus replication. Therefore, these emigrated CD4 T cells represent a novel model of a latent inducible HIV reservoir. Thus, PTX-B and the clinically approved PT-9K/129G are potential antiretroviral agents endowed with immunostimulatory capacity.     

Cooperative inhibition of NF-kappa B and Tat-induced superactivation of human immunodeficiency virus type 1 long terminal repeat.

Human immunodeficiency virus type 1 (HIV-1) long terminal repeat (LTR)-regulated gene expression is stimulated independently by the cellular trans-activator NF-kappa B and the viral protein Tat. Noncytotoxic concentrations of the drug pentoxifylline (PTX) inhibited interaction of NF-kappa B with its motif and the stimulation of HIV-1 LTR-driven gene expression in Jurkat cells. Tat protein (from a cotransfected Tat-expression vector) also induced activation of HIV-1 LTR-driven gene expression. This activation was unaffected by PTX when NF-kappa B sites in the HIV-1 LTR were mutated, suggesting that this drug does not directly influence Tat function, which, however, was inhibited by the Tat-inhibitor Ro 24-7429. Transient reporter gene expression regulated by HIV-1 LTR with wild-type NF-kappa B motifs in the presence of Tat protein was 10- to 60-fold higher than in the presence of either of the trans-activators alone, demonstrating superactivation of HIV-1 LTR by the concerted action of both the trans-activators. Treatment of cells with either PTX or Ro 24-7429 inhibited this superactivation of the HIV-1 LTR. The inhibitory effect of these two drugs in combination, at concentrations that alone did not significantly influence viral promoter activity, was far more than additive. A cooperative action of PTX (NF-kappa B inhibitor) and Ro 24-7429 (Tat inhibitor) on HIV-1 LTR-regulated gene expression is suggested. Concentrations of the drugs that induced maximum inhibition of HIV-1 LTR through their cooperative action are far below cytotoxic levels. Thus, the combination of these two inhibitors could be very effective for anti-HIV therapy.     

CADA, a potential anti-HIV microbicide that specifically targets the cellular CD4 receptor

The cyclotriazadisulfonamide (CADA) compounds are a new class of specific CD4-targeted HIV entry inhibitors. The in vitro anti-HIV activity of CADA was shown to correlate with its ability to specifically downmodulate cell surface expression of the CD4 receptor in human cells. Here, we evaluated its potential as an anti-HIV microbicide. CADA exerted a clear CD4 receptor downregulating effect in dendritic cells (DC) and subsequently inhibited HIV-1(BaL) replication in DC/T cell co-cultures. The compound proved to be active against a variety of clinical isolates belonging to the HIV-1 subtypes A, B, C, D, F, G, H, AE and O. Furthermore, it prevented human T cells from being infected with the laboratory-adapted strains X4 HIV-1(NL4.3) and R5 SIV(mac251). Flow cytometric analysis demonstrated a significant and dose-dependent downregulation of CD4 on macaque PBMCs. In addition, the compound exerted a marked anti-SIV(mac251) activity in these cells from simian origin. The combination of CADA with cellulose acetate phthalate (CAP) resulted in a synergistic inhibition of HIV-1 and SIV infection. Finally, gel formulated CADA proved to preserve the CD4 downmodulating and antiviral activity of this compound when formulated as a microbicide gel. Thus, our data suggest that CADA may have potential as a broad-spectrum anti-HIV microbicide drug candidate. The preservation of the activity of gel formulated CADA will make it now feasible for testing this unique entry inhibitor in non-human primates, not only as a single drug but also in a synergistic conjunction with other anti-HIV compounds.     

Pertussis toxin enhances human immunodeficiency virus type 1 replication

Pertussis toxin (PTX) has been used as a reagent to identify involvement of the G protein-mediated signal transduction pathway. In this study, we found that PTX enhanced HIV-1 replication in acute infection systems at a high dose (1-10 microg/ml) in vitro. PTX treatment enhanced the infectivity of HIV-1-based pseudovirus enveloped with HIV-1 or amphotropic murine leukemia virus (A-MuLV), but not with vesicular stomatitis virus (VSV). This high dose of PTX treatment did not affect HIV-1 gene expression. These data suggested that the effect was virus envelope dependent and that PTX acted on an early stage of viral infection. Treatment with B-oligomer, a nonenzymatic subunit of PTX, mimicked this enhancing effect of PTX. However, desialylation of viral and cellular surface glycoproteins, which are receptors for B-oligomer, did not affect the augmentation induced by PTX. These results indicate that the enhancement of HIV-1 replication is mediated through an unknown biological function of B-oligomer.     

Cyanovirin-N inhibits AIDS virus infections in vaginal transmission models

The cyanobacterial protein cyanovirin-N (CV-N) potently inactivates diverse strains of HIV-1 and other lentiviruses due to irreversible binding of CV-N to the viral envelope glycoprotein gp120. In this study, we show that recombinant CV-N effectively blocks HIV-1(Ba-L) infection of human ectocervical explants. Furthermore, we demonstrate the in vivo efficacy of CV-N gel in a vaginal challenge model by exposing CV-N-treated female macaques (Macaca fascicularis) to a pathogenic chimeric SIV/HIV-1 virus, SHIV89.6P. All of the placebo-treated and untreated control macaques (8 of 8) became infected. In contrast, 15 of 18 CV-N-treated macaques showed no evidence of SHIV infection. Further, CV-N produced no cytotoxic or clinical adverse effects in either the in vitro or in vivo model systems. Together these studies suggest that CV-N is a good candidate for testing in humans as an anti-HIV topical microbicide.     

An 85-aa segment of the GB virus type C NS5A phosphoprotein inhibits HIV-1 replication in CD4+ Jurkat T cells

GB virus type C (GBV-C) is an apparently nonpathogenic virus that replicates in T and B lymphocytes and is a common cause of persistent human infection. Among HIV-1-infected individuals, persistent coinfection with GBV-C is associated with prolonged survival, and infection of blood mononuclear cells or CD4+ T cells with GBV-C and HIV in vitro results in significantly reduced HIV-1 replication. To date, the viral protein(s) that lead to HIV inhibition have not been identified. The GBV-C nonstructural phosphoprotein (NS5A) is predicted to have pleotropic effects on cells, including interactions with the IFN-induced dsRNA-activated protein kinase (PKR). We studied GBV-C NS5A to determine whether it is involved in inhibition of HIV replication. GBV-C NS5A protein from an isolate that was cleared by IFN therapy did not inhibit PKR, whereas NS5A from an isolate that was not cleared by IFN-inhibited PKR function in a yeast genetic system. Both of these GBV-C NS5A proteins were expressed in a CD4+ T cell line (Jurkat), and both induced a potent, dose-dependent inhibition of HIV-1 replication, thus the effect was independent of PKR inhibition. NS5A induced the release of the chemokine SDF-1 and decreased surface expression of the HIV coreceptor CXCR4, potentially explaining the HIV inhibition. Deletion mapping of the NS5A protein found that an 85-aa region between amino acids 152 and 237 inhibits HIV-1 replication. Thus, GBV-C NS5A protein alters the cellular milieu necessary for HIV-1 replication and may provide a previously undescribed therapeutic approach for anti-HIV therapy.     

Inhibition of human immunodeficiency virus 1 (HIV-1) and herpes simplex virus 1 (HSV-1) infectivity with a broad range of lectins.

Five lectins with specificity for N- and O-linked oligosaccharides were examined for inhibition of HIV-1 and HSV-1 infectivity in vitro. HIV-1 isolate HTLVIIIB was preincubated with lectin and subsequently inoculated onto MT-4 cells. Lectins specific for N-linked oligosaccharides blocked HIV infection in nanomolar-micromolar concentrations, but no anti-HIV effect was found with the lectin HPA, mainly reacting with O-linked oligosaccharides. HSV-1 infectivity was measured in a plaque reduction assay using Vero cells, and while both N- and O-linked oligosaccharide -specific lectins inhibited HSV-1 infection, the most potent inhibition was found with the lectin HPA. These results indicate that lectins may have a broad antiviral effect on enveloped viruses only limited by types of oligosaccharides present on individual viruses.     

Human immunodeficiency virus type 1 (HIV-1) inhibition, DNA-binding, RNA-binding, and ribosome inactivation activities in the N-terminal segments of the plant anti-HIV protein GAP31.

GAP31 (gelonium anti-HIV protein of 31 kDa) is an anti-HIV protein which we have identified and purified from a medicinal plant, Gelonium multiflorum. It is capable of inhibiting HIV-1 infection and replication. GAP31 also exhibits DNA topoisomerase inhibitor activity and RNA N-glycosidase activity. The ability of GAP31 to interrupt both DNA and RNA functions may be related to its multiple antiviral actions. To define the roles of these activities in the anti-HIV action of GAP31, a series of peptides corresponding to the N-terminal segment of GAP31 were synthesized and assayed for the aforementioned activities of the parent molecule. A 33-aa segment (KGATYITYVNFLNELRVKTKPEGNSHGIPSLRK) designated as K10-K42 is the shortest peptide necessary and sufficient for HIV-1 inhibition, DNA and RNA binding, and ribosome inactivation. The peptides were 2-5 orders of magnitude less active than GAP31. Truncation of 19 aa from the C terminus of K10-K42 resulted in the loss of all of these activities. On the other hand, deletion of N-terminal residues to give E23-K42 did not alter ribosome-inactivation activity but eliminated the other activities. These findings permit identification of a 7-aa sequence, KGATYIT, at the N terminus of K10-K42 that is critical for DNA binding and RNA binding, whereas a 9-aa sequence, SHGIPSLRK, at the C terminus is important to ribosome inactivation. Both regions contribute to anti-HIV activity. Histidine at position 35 is critical for all of these activities. The disparity of sequence requirements for inhibition of HIV infection and replication and for ribosome-inactivation activity suggests that the anti-HIV activity of most ribosome-inactivating proteins may not be the result of N-glycosidase activity alone. Mapping the minimal domain of GAP31 offers insights into the rational design of molecular mimetics of anti-HIV drugs.     

Infection of vaginal and colonic epithelial cells by the human immunodeficiency virus type 1 is neutralized by antibodies raised against conserved epitopes in the envelope glycoprotein gp120.

The rectal and genital tract mucosae are considered to be major sites of entry for the human immunodeficiency virus (HIV) during sexual contact. We now demonstrate that vaginal epithelial cells can be infected by HIV type 1 (HIV-1) via a mechanism similar to that described for neuroglial cells and, more recently, for colorectal epithelial cells, involving initial interaction of the HIV-1 envelope glycoprotein gp120 with a cell-surface glycosphingolipid (sulfated lactosylceramide). A hyperimmune serum against gp120 was able to neutralize HIV-1 infection of vaginal epithelial cells. Site-directed immunization was employed to identify sites on gp120 recognized by antibodies neutralizing HIV-1 infection of vaginal and colonic epithelial cells. Hyperimmune sera were raised in monkeys against a series of 40 overlapping synthetic peptides covering the entire sequence of HIV-1 (HTLV-IIIB) gp120. Antisera raised against five synthetic peptides, corresponding to three relatively conserved regions and to the hypervariable region (V3 loop), efficiently neutralized HIV-1 infection of human vaginal epithelial cells in vitro. Similar results were obtained with the colonic cells. Hyperimmune sera to all five peptides have been shown earlier to neutralize HIV-1 infectivity in CD4+ T cells. These results have obvious implications for the design of mucosal subunit vaccines against sexually transmitted HIV-1 infections.     

河北省某县有偿献血丙型肝炎病毒感染者血清抗-HIV检测分析

目的：调查并分析河北省某县有偿献血员中丙型肝炎病毒(HCV)感染者艾滋病病毒Ⅰ型(HIV-1)感染情况。方法：采用实时定量聚合酶链反应(PCR)和酶联免疫吸附试验(ELISA)法等检测了抗-HCV、HCV核糖核酸(RNA)和抗-HIV。结果：共检测109例受HCV感染的献血员，其中ALT异常者为38．53％(42／109)，抗-HCV阳性率为95．41％(104／109)，HCV RNA阳性率为70.64％(77／109)，抗-HIV阳性率为0(0／109)。结论：在该县有偿献血HCV感染者中HIV-1感染率为0，HCV感染者中HIV-1感染存在地区差异，并非所有地区HCV感染者HIV感染率均高。     

Evaluation of an enzyme immunoassay for the combined detection of antibodies to HIV-1, HIV-2, HTLV-I and HTLV-II.

OBJECTIVE: To assess the sensitivity and specificity of a newly-developed assay (Bioelisa HIV-1 + 2, HTLV-1 + 2) for the simultaneous detection of HIV-1, HIV-2, HTLV-I and HTLV-II antibodies in human serum or plasma specimens. METHODS: A panel of 775 well characterized serum or plasma samples was studied. This included samples confirmed to contain antibodies to HIV-1 (n = 46), HIV-2 (n = 19), HTLV-I (n = 49) and HTLV-II (n = 12), samples containing low titres of anti-HIV antibody (n = 14) and samples collected during HIV seroconversion (n = 36). Eighty-three sera samples which were reactive in one or more HIV or HTLV screening assays, but which could not be confirmed to contain anti-HIV-1/2 or anti-HTLV-I/II antibodies, were also examined. RESULTS: Excluding the seroconversion samples and those selected on the basis of false reactivity in other screening assays, the Bioelisa kit had a sensitivity of 100% for antibody to all four viruses and a specificity of 98.8%. The ability of the kit to detect anti-HIV during seroconversion was similar to that of several other synthetic HIV-antigen-based screening kits currently in use. CONCLUSIONS: Our findings indicate that the Bioelisa kit is sufficiently accurate to screen for both HIV and HTLV infections and that it warrants larger scale trials. Its use might allow blood donor screening for HTLV infection to be introduced more widely at modest extra cost [corrected].     

Inhibition of the integrase of human immunodeficiency virus (HIV) type 1 by anti-HIV plant proteins MAP30 and GAP31.

MAP30 (Momordica anti-HIV protein of 30 kDa) and GAP31 (Gelonium anti-HIV protein of 31 kDa) are anti-HIV plant proteins that we have identified, purified, and cloned from the medicinal plants Momordica charantia and Gelonium multiflorum. These antiviral agents are capable of inhibiting infection of HIV type 1 (HIV-1) in T lymphocytes and monocytes as well as replication of the virus in already-infected cells. They are not toxic to normal uninfected cells because they are unable to enter healthy cells. MAP30 and GAP31 also possess an N-glycosidase activity on 28S ribosomal RNA and a topological activity on plasmid and viral DNAs including HIV-1 long terminal repeats (LTRs). LTRs are essential sites for integration of viral DNA into the host genome by viral integrase. We therefore investigated the effect of MAP30 and GAP31 on HIV-1 integrase. We report that both of these antiviral agents exhibit dose-dependent inhibition of HIV-1 integrase. Inhibition was observed in all of the three specific reactions catalyzed by the integrase, namely, 3' processing (specific cleavage of the dinucleotide GT from the viral substrate), strand transfer (integration), and "disintegration" (the reversal of strand transfer). Inhibition was studied by using oligonucleotide substrates with sequences corresponding to the U3 and U5 regions of HIV LTR. In the presence of 20 ng of viral substrate, 50 ng of target substrate, and 4 microM integrase, total inhibition was achieved at equimolar concentrations of the integrase and the antiviral proteins, with EC50 values of about 1 microM. Integration of viral DNA into the host chromosome is a vital step in the replicative cycle of retroviruses, including the AIDS virus. The inhibition of HIV-1 integrase by MAP30 and GAP31 suggests that impediment of viral DNA integration may play a key role in the anti-HIV activity of these plant proteins.     

Infection of hematopoietic progenitor cells by HIV-1 subtype C, and its association with anemia in southern Africa

Reports from southern Africa, an area in which human immunodeficiency virus type 1 (HIV-1) infection is caused almost exclusively by subtype C (HIV-1C), have shown increased rates of anemia in HIV-infected populations compared with similar acquired immunodeficiency syndrome (AIDS) patients in the United States, an area predominantly infected with subtype B (HIV-1B). Recent findings by our group demonstrated a direct association between HIV-1 infection and hematopoietic progenitor cell health in Botswana. Therefore, using a single-colony infection assay and quantitative proviral analysis, we examined whether HIV-1C could infect hematopoietic progenitor cells (HPCs) and whether this phenotype was associated with the higher rates of anemia found in southern Africa. The results show that a significant number of HIV-1C, but not HIV-1B, isolates can infect HPCs in vitro (P < .05). In addition, a portion of HIV-1C-positive Africans had infected progenitor cell populations in vivo, which was associated with higher rates of anemia in these patients (P < .05). This represents a difference in cell tropism between 2 geographically separate and distinct HIV-1 subtypes. The association of this hematotropic phenotype with higher rates of anemia should be considered when examining anti-HIV drug treatment regimens in HIV-1C-predominant areas, such as southern Africa.     

Lysozyme and RNases as anti-HIV components in beta-core preparations of human chorionic gonadotropin

Human chorionic gonadotropin (hCG) preparations contain activity against HIV type 1 (HIV-1). However, there has been controversy about whether some biological activities of hCG beta-subunit (hCGbeta) preparations are caused by the beta-subunit itself or other proteins present in the preparations. We report here the purification, characterization, and identification of three enzymes with anti-HIV activity present in the beta-core fraction of hCGbeta prepared from the urine of pregnant women. The N-terminal amino acid sequence of one protein is identical to human urinary lysozyme C, and those of the other two are identical to human RNase A and urinary RNase U. We thus refer to these proteins as AVL (antiviral lysozyme) and AVR (antiviral RNases). In addition to HIV-1 inhibition, AVL is capable of lysing Micrococcus lysodeikticus. AVR digests a variety of RNA substrates, including RNA from HIV-1-infected cells. We also find that lysozyme from chicken egg white, human milk, and human neutrophils and RNase A from bovine pancreas possess activity against HIV-1. These findings may offer additional strategies for the treatment of HIV-1 infection.     

Short communication: Increased expression of secretory leukocyte protease inhibitor in oral mucosa of Colombian HIV type 1-exposed seronegative individuals

The exposure to human immunodeficiency virus type 1 (HIV-1) does not always result in infection. Indeed, there are individuals who have been repeatedly exposed to HIV-1 but do not exhibit clinical or serological evidence of infection; they are known as HIV-exposed seronegative individuals (HESN). To determine if secretory leukocyte protease inhibitor (SLPI), a soluble factor secreted by epithelial cells lining mucosal surfaces that showed anti-HIV activity in vitro, was associated with natural resistance to HIV infection, we measured by real time RT-PCR the expression of SLPI in oral mucosa of a cohort of Colombian HESN, in chronically HIV-1-infected individuals and in healthy controls. The HESN expressed significantly higher levels of SLPI mRNA than healthy controls (p=0.033) and chronically infected subjects (p=0.011). These findings suggest an association between SLPI expression and the natural resistance to HIV-1 infection exhibited by our HESN cohort.     

Inhibition of hematopoietic progenitor cell proliferation by ethanol in human immunodeficiency virus type 1 tat-expressing transgenic mice

BACKGROUND: A number of hematological abnormalities are associated with both human immunodeficiency virus type 1 (HIV-1) infection and alcohol abuse. There is little information on how alcohol abuse might further influence the survival and growth of hematopoietic progenitors in HIV-infected individuals in the presence of immune system abnormalities and anti-HIV drugs. Because there is evidence that viral transactivator Tat itself can induce hematopoietic suppression, in this study we examined the role of ethanol as a cofactor in transgenic mice that expressed HIV-1 Tat protein. METHODS: Tat transgenic mice and nontransgenic littermates were given ethanol (20% v/v) and the anti-HIV drug 3'-azido-3'-deoxythymidine (AZT; 1 mg/ml) in drinking water. Immunosuppression in mice was induced by weekly intraperitoneal injections of anti-CD4 antibody. Hematopoiesis was examined by erythroid colony forming unit (CFU-E) and granulocyte/macrophage colony-forming unit (CFU-GM) assays of the bone marrow progenitor cells. RESULTS: Administration of ethanol for 7 weeks resulted in a 50% decrease in the proliferative capacity of CFU-E- and CFU-GM-derived progenitors from transgenic mice compared with that of ethanol-treated nontransgenic controls. Similar decreases also were observed in transgenic mice treated with AZT or a combination of AZT and ethanol. Furthermore, ethanol and AZT were significantly more toxic to the granulopoietic progenitors (40-50% inhibition) than to the erythropoietic progenitors (10-20% inhibition) in Tat transgenic mice. Although a 10 day exposure of Tat transgenic and nontransgenic mice to a combination of ethanol and AZT had no suppressive effect on the erythropoietic and granulopoietic progenitor cells, there was a marked decrease (40-60%) in CFU-GM in mice made immunodeficient by CD4+ T-lymphocyte depletion. The ethanol-treated Tat transgenic mice but not the nontransgenic litter-mates also showed a significant decrease (25%) in CFU-GM. CONCLUSION: Our in vivo study strongly suggests that ethanol ingestion in HIV-1-infected individuals, particularly those on antiretroviral drugs, might increase bone marrow toxicity and contribute to HIV-1-associated hematopoietic impairment.     

Inhibition of HIV-1 replication in human lymphoid tissues ex vivo by measles virus

Human immunodeficiency virus (HIV) type 1 replication and disease progression are enhanced by various pathogens in coinfected individuals. However, acute infection with measles virus (MV) has been found to suppress HIV-1 replication in coinfected children. We investigated the mechanisms of this phenomenon using human lymphoid tissues coinfected ex vivo with HIV-1 and MV. MV inhibited both CXCR4-tropic (X4) and CCR5-tropic (R5) HIV-1, but the inhibitory effect was particularly profound for R5 virus, which transmits infection and dominates the early stages of HIV-1 disease. MV inhibits the replication of R5 HIV-1 in coinfected tissues by up-regulation of the CC chemokine RANTES, a well-known inhibitor of R5 HIV-1 infection, and this up-regulation is augmented in tissues coinfected with R5 HIV-1. Deciphering the molecular mechanisms by which MV and other pathogens alter local cytokine/chemokine networks and cause tissue microenvironments to become detrimental to HIV-1 may significantly contribute to the development of effective anti-HIV therapies.