Vaginal microbicides with activity towards organisms that cause sexually transmitted infections have been proposed as a strategy to reduce transmission. Small-molecule inhibitors of Chlamydia trachomatis serovar D belonging to the class of salicylidene acylhydrazides (INPs) have been shown to work through a mechanism that involves iron restriction. Expanding on this work, ten INPs were tested against a lymphogranuloma venereum strain of C. trachomatis serovar L2, Neisseria gonorrhoeae, and hydrogen peroxide-producing Lactobacillus crispatus and Lactobacillus jensenii. Seven INPs had minimal inhibitory concentrations (MICs) and minimal bactericidal concentrations of <50 µM towards C. trachomatis L2. Three INPs had an MIC <12.5 µM against N. gonorrhoeae. Inhibition by was reversed by iron, holo-transferrin and holo-lactoferrin but not by the iron-poor forms of these compounds. The compounds exhibited no bactericidal activity toward Lactobacillus. The INPs were not cytotoxic to HeLa 229 cells. When INP 0341 was tested in a mouse model of a Chlamydia vaginal infection there was a significant reduction in the number of mice shedding C. trachomatis up to 4 days after infection (P < 0.01). In summary, select INPs are promising vaginal microbicide candidates as they inhibit the growth of two common sexually transmitted organisms in vitro, are active in a mouse model against C. trachomatis, are not cytotoxic and do not inhibit organisms that compose the normal vaginal flora.
Vaginal microbicide; Sexually transmitted infections; Chlamydia trachomatis; LGV; Neisseria gonorrhoeae
A collection of nine salicylidene acylhydrazide compounds were tested for their ability to inhibit the activity of virulence-associated type III secretion systems (T3SSs) in Salmonella enterica serovar Typhimurium. The compounds strongly affected Salmonella pathogenicity island 1 (SPI1) T3SS-mediated invasion of epithelial cells and in vitro secretion of SPI1 invasion-associated effector proteins. The use of a SPI1 effector β-lactamase fusion protein implicated intracellular entrapment of the protein construct upon application of a salicylidene acylhydrazide, whereas the use of chromosomal transcriptional gene fusions revealed a compound-mediated transcriptional silencing of SPI1. Salicylidene acylhydrazides also affected intracellular bacterial replication in murine macrophage-like cells and blocked the transport of an epitope-tagged SPI2 effector protein. Two of the compounds significantly inhibited bacterial motility and expression of extracellular flagellin. We conclude that salicylidene acylhydrazides affect bacterial T3SS activity in S. enterica and hence could be used as lead substances when designing specific inhibitors of bacterial T3SSs in order to pharmaceutically intervene with bacterial virulence.
Salicylidene acylhydrazides belong to a class of compounds shown to inhibit bacterial type III secretion (T3S) in pathogenic Gram-negative bacteria. This class of compounds also inhibits growth and replication of Chlamydiae, strict intracellular bacteria that possess a T3S system. In this study a library of 58 salicylidene acylhydrazides was screened to identify inhibitors of Chlamydia growth. Compounds inhibiting growth of both Chlamydia trachomatis and Chlamydophila pneumoniae were tested for cell toxicity and seven compounds were selected for preliminary pharmacokinetic analysis in mice using cassette dosing. Two compounds, ME0177 and ME0192, were further investigated by individual pharmacokinetic analysis. Compound ME0177 had a relatively high peak plasma concentration (Cmax) and area under curve and therefore may be considered for systemic treatment of Chlamydia infections. The other compound, ME0192, had poor pharmacokinetic properties but the highest anti-chlamydial activity in vitro and therefore was tested for topical treatment in a mouse vaginal infection model. ME0192 administered vaginally significantly reduced the infectious burden of C. trachomatis and the number of infected mice.
Chlamydophila pneumoniae; Chlamydia trachomatis; pre-clinical pharmacokinetics; type III secretion inhibitor; vaginal microbicide; virulence inhibitor
The salicylidene acylhydrazide INP0341 inhibits growth of Chlamydia in HeLa cells, has negligible cell toxicity, and does not inhibit the growth of lactobacilli. The antichlamydial activity of INP0341 was retained when tested in vaginal and semen simulants. Vaginal tissue from INP0341-treated mice appeared similar to control sham-treated mice. To determine whether INP0341 can protect mice from a vaginal challenge, C3H/HeJ mice were either sham or INP0341 treated intravaginally pre- and postinoculation with 5 × 102 inclusion-forming units (IFUs) of Chlamydia trachomatis serovar D. Vaginal cultures taken over a month-long period showed a significant difference in the number of control mice that were culture positive versus the number in the INP0341-treated group, 100% (25/25) and 31% (8/26), respectively (P < .05). The quantity of IFUs shed and antibody titers to Chlamydia were significantly higher for the control group (P < .05). In summary, INP0341 is a promising compound to be considered for formulation as a vaginal microbicide.
A class of anti-virulence compounds, the salicylidene acylhydrazides, has been widely reported to block the function of the type three secretion system of several Gram-negative pathogens by a previously unknown mechanism. In this work we provide the first identification of bacterial proteins that are targeted by this group of compounds. We provide evidence that their mode of action is likely to result from a synergistic effect arising from a perturbation of the function of several conserved proteins. We also examine the contribution of selected target proteins to the pathogenicity of Yersinia pseudotuberculosis and to expression of virulence genes in Escherichia coli O157.
Bacteria; Bacterial Genetics; Gene Regulation; Protein Drug Interactions; Protein Secretion; Protein Targeting; Transcription
Type III secretion systems (T3SSs) are essential virulence devices for many gram-negative bacteria that are pathogenic for plants, animals, and humans. They serve to translocate virulence effector proteins directly into eukaryotic host cells. T3SSs are composed of a large cytoplasmic bulb and a transmembrane region into which a needle is embedded, protruding above the bacterial surface. The emerging antibiotic resistance of bacterial pathogens urges the development of novel strategies to fight bacterial infections. Therapeutics that rather than kill bacteria only attenuate their virulence may reduce the frequency or progress of resistance emergence. Recently, a group of salicylidene acylhydrazides were identified as inhibitors of T3SSs in Yersinia, Chlamydia, and Salmonella species. Here we show that these are also effective on the T3SS of Shigella flexneri, where they block all related forms of protein secretion so far known, as well as the epithelial cell invasion and induction of macrophage apoptosis usually demonstrated by this bacterium. Furthermore, we show the first evidence for the detrimental effect of these compounds on T3SS needle assembly, as demonstrated by increased numbers of T3S apparatuses without needles or with shorter needles. Therefore, the compounds generate a phenocopy of T3SS export apparatus mutants but with incomplete penetrance. We discuss why this would be sufficient to almost completely block the later secretion of effector proteins and how this begins to narrow the search for the molecular target of these compounds.
Salicylidene acylhydrazides (SAHs) inhibit the type III secretion system (T3S) of Yersinia and other Gram-negative bacteria. In addition, SAHs restrict the growth and development of Chlamydia species. However, since the inhibition of Chlamydia growth by SAH is suppressed by the addition of excess iron and since SAHs have an iron-chelating capacity, their role as specific T3S inhibitors is unclear. We investigated here whether SAHs exhibit a function on C. trachomatis that goes beyond iron chelation. We found that the iron-saturated SAH INP0341 (IS-INP0341) specifically affects C. trachomatis infectivity with reduced generation of infectious elementary body (EB) progeny. Selection and isolation of spontaneous SAH-resistant mutant strains revealed that mutations in hemG suppressed the reduced infectivity caused by IS-INP0341 treatment. Structural modeling of C. trachomatis HemG predicts that the acquired mutations are located in the active site of the enzyme, suggesting that IS-INP0341 inhibits this domain of HemG and that protoporphyrinogen oxidase (HemG) and heme metabolism are important for C. trachomatis infectivity.
Thiol peroxidase, Tpx, has been shown to be a target protein of the salicylidene acylhydrazide class of antivirulence compounds. In this study we present the crystal structures of Tpx from Y. pseudotuberculosis (ypTpx) in the oxidised and reduced states, together with the structure of the C61S mutant. The structures solved are consistent with previously solved atypical 2-Cys thiol peroxidases, including that for “forced” reduced states using the C61S mutant. In addition, by investigating the solution structure of ypTpx using small angle X-ray scattering (SAXS), we have confirmed that reduced state ypTpx in solution is a homodimer. The solution structure also reveals flexibility around the dimer interface. Notably, the conformational changes observed between the redox states at the catalytic triad and at the dimer interface have implications for substrate and inhibitor binding. The structural data were used to model the binding of two salicylidene acylhydrazide compounds to the oxidised structure of ypTpx. Overall, the study provides insights into the binding of the salicylidene acylhydrazides to ypTpx, aiding our long-term strategy to understand the mode of action of this class of compounds.
Salicylidene acylhydrazides identified as inhibitors of virulence-mediating type III secretion systems (T3SSs) potentially target their inner membrane export apparatus. They also lead to inhibition of flagellar T3SS-mediated swimming motility in Salmonella enterica serovar. Typhimurium. We show that INP0404 and INP0405 act by reducing the number of flagella/cell. These molecules still inhibit motility of a Salmonella ΔfliH-fliI-fliJ/flhB(P28T) strain, which lacks three soluble components of the flagellar T3S apparatus, suggesting that they are not the target of this drug family. We implemented a genetic screen to search for the inhibitors' molecular target(s) using motility assays in the ΔfliH-fliI/flhB(P28T) background. Both mutants identified were more motile than the background strain in the absence of the drugs, although HM18 was considerably more so. HM18 was more motile than its parent strain in the presence of both drugs while DI15 was only insensitive to INP0405. HM18 was hypermotile due to hyperflagellation, whereas DI15 was not hyperflagellated. HM18 was also resistant to a growth defect induced by high concentrations of the drugs. Whole-genome resequencing of HM18 indicated two alterations within protein coding regions, including one within atpB, which encodes the inner membrane a-subunit of the FOF1-ATP synthase. Reverse genetics indicated that the alteration in atpB was responsible for all of HM18's phenotypes. Genome sequencing of DI15 uncovered a single A562P mutation within a gene encoding the flagellar inner membrane protein FlhA, the direct role of which in mediating drug insensitivity could not be confirmed. We discuss the implications of these findings in terms of T3SS export apparatus function and drug target identification.
Small chemical compounds which target chemokine receptors have been developed against human immunodeficiency virus type 1 (HIV-1) and are under investigation for use as anti-HIV-1 microbicides. In addition, monoclonal antibodies (mAbs) against chemokine receptors have also been shown to have anti-HIV-1 activities. The objective of the present study was to screen a panel of three anti-CXCR4 specific monoclonal antibodies (mAbs) for their ability to block the HIV-1 infection using in vitro activated primary peripheral blood mononuclear cells (PBMCs).
PBMCs from normal donors were pre-activated with anti-CD3 and anti-CD28 mAbs for 1 day, and aliquots were infected with a low dose of CCR5-tropic (R5), CXCR4 tropic (X4) or dual tropic (X4R5) HIV-1 isolates and cultured in the presence of a panel of anti-CXCR4 mAbs. The panel included clones A145 mAb against the N-terminus, A120 mAb against a conformational epitope consisting of extracellular loops (ECL)1 and ECL2, and A80 mAb against ECL3 of CXCR4. Among these mAbs, the A120 mAb showed the most potent inhibition of infection, by not only X4 but surprisingly also R5 and X4R5 HIV-1. The inhibition of R5 HIV-1 was postulated to result from the novel ability of the A120 mAb to induce the levels of the CCR5-binding β-chemokines MIP-1α, MIP-1β and/or RANTES, and the down modulation of CCR5 expression on activated CD4+ T cells. Neutralizing anti-MIP-1α mAb significantly reversed the inhibitory effect of the A120 mAb on R5 HIV-1 infection.
The data described herein have identified a unique epitope of CXCR4 whose ligation not only directly inhibits X4 HIV-1, but also indirectly inhibits R5 HIV-1 infection by inducing higher levels of natural CCR5 ligands.
OBJECTIVE: To investigate the in vitro microbicidal and cytocidal potency of monocaprin dissolved in pharmaceutical hydrogel formulations and to evaluate their potential use as vaginal microbicides against sexually transmitted pathogens such as herpes simplex virus type 2 (HSV-2), human immunodeficiency virus type 1 (HIV-1), Chlamydia trachomatis, and Neisseria gonorrhoeae. METHODS: Gel formulations were mixed with equal volumes of virus/bacteria suspensions in culture medium and incubated for 1 and 5 minutes. The reduction in virus/bacteria titre was used as a measure of microbicidal activity. Similarly, gels were mixed with human semen to study their effect on leucocytes. The toxicity of the gels was tested in rabbits by the standard vaginal irritation test. RESULTS: Gels containing 20 mM of monocaprin caused a greater than 100,000-fold inactivation of HSV-2 and Neisseria in 1 minute and of Chlamydia in 5 minutes. Similarly, the gels caused a greater than 10,000-fold inactivation of HIV-1 in semen in 1 minute. They caused more than a 10,000-fold reduction in the number of viable leucocytes in semen in 1 minute. No toxic effect on the vaginal mucosa of rabbits was observed after daily exposure for 10 days. CONCLUSIONS: Hydrogels containing monocaprin are potent inactivators of sexually transmitted viruses and bacteria in vitro. This simple lipid seems to be a feasible choice as a mucosal microbicide for prevention of sexually transmitted infections. It is a natural compound found in certain foodstuffs such as milk and is therefore unlikely to cause harmful side effects in the concentrations used.
An effective anti-human immunodeficiency virus-1 (HIV-1) microbicide should exert its action in the absence of causing aberrant activation of topical immunity that will increase the risk of HIV acquisition. In the present study, we demonstrated that the vaginal application of cellulose sulfate (CS) gel induced topical mucosal inflammatory responses; the addition of minocycline to CS gel could significantly attenuate the inflammation in a mice model. The combined gel of CS plus minocycline not only reduced the production of inflammatory cytokines in cervicovaginal lavages (CVLs), also down-regulated the activation of CD4+ T cells and the recruitment of other immune cells including HIV target cells into vaginal tissues. Furthermore, an In vitro HIV-1 pseudovirus infection inhibition assay showed that the combined gel decreased the infection efficacy of different subtypes of HIV-1 pseudoviruses compared with that of CS gel alone. These results implicate that minocycline could be integrated into microbicide formulation to suppress the aberrant activation of topical mucosal immunity and enhance the safety profile during the application of microbicides.
In the absence of a fully effective human immunodeficiency virus (HIV) vaccine, topical microbicides represent an important strategy for preventing the transmission of HIV through sexual intercourse, the predominant mode of HIV transmission worldwide. Although a comprehensive understanding of HIV transmission has not yet emerged in the microbicide field, it is likely the result of rapid infection of monocyte-derived cells in the vaginal mucosa by CCR5-tropic viruses. Inhibition of HIV transmission requires agents that prevent entry, fusion, reverse transcription, or other preintegrative replication events or agents which directly inactivate HIV or modulate the target cells to render them uninfectible. In vitro assays typically used to evaluate the ability of a microbicide to prevent virus transmission use epithelial or human osteosarcoma-derived cells or immune cells more relevant to the development of anti-HIV therapeutic agents and quantify virus production at short time intervals following infection. We have developed a microbicide transmission and sterilization assay (MTSA) to more sensitively and quantitatively evaluate virus transmission in cell culture in the presence of microbicidal compounds. Results obtained with the MTSA demonstrate that the inhibitory capacity of microbicides is often overestimated in short-term transmission inhibition assays, while some compounds yield equivalent inhibitory results, indicating a biological relevance for the MTSA-based evaluations to identify superior potent microbicides. The MTSA defines the concentration of the microbicide required to totally suppress the transmission of virus in cell culture and may thus help define the effective concentration of the microbicide required in a formulated microbicide product.
The development of a topical microbicide blocking the sexual transmission of HIV-1 is urgently needed to control the global HIV/AIDS pandemic. The actinomycete-derived lectin actinohivin (AH) is highly specific to a cluster of high-mannose-type glycans uniquely found on the viral envelope (Env). Here, we evaluated AH's candidacy toward a microbicide in terms of in vitro anti-HIV-1 activity, potential side effects, and recombinant producibility. Two validated assay systems based on human peripheral blood mononuclear cell (hPBMC) infection with primary isolates and TZM-bl cell infection with Env-pseudotyped viruses were employed to characterize AH's anti-HIV-1 activity. In hPMBCs, AH exhibited nanomolar neutralizing activity against primary viruses with diverse cellular tropisms, but did not cause mitogenicity or cytotoxicity that are often associated with other anti-HIV lectins. In the TZM-bl-based assay, AH showed broad anti-HIV-1 activity against clinically-relevant, mucosally transmitting strains of clades B and C. By contrast, clade A viruses showed strong resistance to AH. Correlation analysis suggested that HIV-1′s AH susceptibility is significantly linked to the N-glycans at the Env C2 and V4 regions. For recombinant (r)AH expression, we evaluated a tobacco mosaic virus-based system in Nicotiana benthamiana plants as a means to facilitate molecular engineering and cost-effective mass production. Biochemical analysis and an Env-mediated syncytium formation assay demonstrated high-level expression of functional rAH within six days. Taken together, our study revealed AH's cross-clade anti-HIV-1 activity, apparent lack of side effects common to lectins, and robust producibility using plant biotechnology. These findings justify further efforts to develop rAH toward a candidate HIV-1 microbicide.
In the absence of an effective vaccine against the human immunodeficiency virus (HIV), topical microbicides to prevent the sexual transmission of HIV represent an important strategy to prevent the continued spread of infection. The recent trend in the development of new microbicide candidates includes the utilization of FDA-approved therapeutic drugs that target the early stages of the HIV life cycle, including entry inhibitors and reverse transcriptase inhibitors. We have investigated 12 pyrimidinedione compounds with potent HIV activities and their abilities to inhibit both virus entry and reverse transcription, in an effort to determine a lead microbicide for product development. The candidate compounds were evaluated for efficacy against subtype B, C, and E clinical virus strains in fresh human peripheral blood mononuclear cells and against CCR5-tropic virus strains in both monocyte-macrophages and dendritic cells. Microbicide-specific biological assays and toxicity evaluations were also performed in a variety of established and fresh human cells as well as against Lactobacillus strains common to the vaginal environment. These evaluations resulted in the identification of congeners with cyclopropyl and cyclobutyl substituents at the N-1 of the pyrimidinedione as the most active molecules in the structure-activity relationship series. The pyrimidinediones represent excellent microbicide candidates in light of their significantly high efficacies against HIV-1 (subnanomolar concentration range), potencies (therapeutic index, >1 million), solubility profiles, and dual mechanism of antiviral action that includes two early steps of virus replication prior to the integration of the virus that are considered most important for microbicidal activity.
Topical microbicides may prove to be an important strategy for preventing human immunodeficiency virus type 1 (HIV-1) transmission. We examined the safety and efficacy of sequence-nonspecific phosphorothioate 2′ deoxyribose oligomers as potential novel microbicides. A short, 13-mer poly(T) phosphorothioate oligodeoxynucleotide (OPB-T) significantly inhibited infection of primary peripheral blood mononuclear cells (PBMC) by high-titer HIV-1Ba-L and simian immunodeficiency virus mac251 (SIVmac251). Continuous exposure of human vaginal and foreskin tissue explants to OPB-T showed no toxicity. An abasic 14-mer phosphorothioate 2′ deoxyribose backbone (PDB) demonstrated enhanced anti-HIV-1 activity relative to OPB-T and other homo-oligodeoxynucleotide analogs. When PDB was used to pretreat HIV-1, PDB was effective against R5 and X4 isolates at a half-maximal inhibitory concentration (IC50) of <1 μM in both PBMC and P4-R5 MAGI cell infections. PDB also reduced HIV-1 infectivity following the binding of virus to target cells. This novel topical microbicide candidate exhibited an excellent in vitro safety profile in human PBMC and endocervical epithelial cells. PDB also retained activity in hydroxyethylcellulose gel at pH 4.4 and after transition to a neutral pH and was stable in this formulation for 30 days at room temperature. Furthermore, the compound displayed potent antiviral activity following incubation with a Lactobacillus strain derived from normal vaginal flora. Most importantly, PDB can inhibit HIV-1-induced alpha interferon production. Phosphorothioate 2′ deoxyribose oligomers may therefore be promising microbicide candidates that inhibit HIV-1 infection and also dampen the inflammation which is critical for the initial spread of the virus.
Human immunodeficiency virus (HIV), causative agent of acquired immunodeficiency syndrome (AIDS), is a global health concern. To control its transmission, safe sex has been proposed as one of the strategies. Microbicides- intravaginal/intrarectal topical formulations of anti-HIV agents have also been proposed to prevent HIV transmission. Microbicides would provide protection by directly inactivating HIV or preventing the attachment, entry or replication of HIV in susceptible target cells as well as their dissemination from target cells present in semen or the host cells lining the vaginal/rectal wall to other migratory cells. Microbicides must be safe, effective following vaginal or rectal administration, and should cause minimal or no genital symptoms or inflammations following long-term repeated usage. However, a safe and efficacious anti-HIV microbicide is not yet available despite the fact that more than 60 candidate agents have been identified to have in vitro activity against HIV, several of which have advanced to clinical testing. Nonetheless, proof-of-concept of microbicides has been established based on the results of recent CAPRISA 004 clinical trials. In this article, the trends and challenges in the development of effective and safe microbicides to combat HIV transmission are reviewed.
HIV; intravaginal; microbicides; prevention
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.
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.
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.
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 microbicides are being investigated in clinical trials and understanding how promising strategies work, coincident with demonstrating efficacy in vivo, is central to advancing new generation microbicides. We evaluated Carraguard® and a new generation Carraguard-based formulation containing the non-nucleoside reverse transcriptase inhibitor (NNRTI) MIV-150 (PC-817). Since dendritic cells (DCs) are believed to be important in HIV transmission, the formulations were tested for the ability to limit DC-driven infection in vitro versus vaginal infection of macaques with RT-SHIV (SIVmac239 bearing HIV reverse transcriptase). Carraguard showed limited activity against cell-free and mature DC-driven RT-SHIV infections and, surprisingly, low doses of Carraguard enhanced infection. However, nanomolar amounts of MIV-150 overcame enhancement and blocked DC-transmitted infection. In contrast, Carraguard impeded infection of immature DCs coincident with DC maturation. Despite this variable activity in vitro, Carraguard and PC-817 prevented vaginal transmission of RT-SHIV when applied 30 min prior to challenge. PC-817 appeared no more effective than Carraguard in vivo, due to the limited activity of a single dose of MIV-150 and the dominant barrier effect of Carraguard. However, 3 doses of MIV-150 in placebo gel at and around challenge limited vaginal infection, demonstrating the potential activity of a topically applied NNRTI. These data demonstrate discordant observations when comparing in vitro and in vivo efficacy of Carraguard-based microbicides, highlighting the difficulties in testing putative anti-viral strategies in vitro to predict in vivo activity. This work also underscores the potential of Carraguard-based formulations for the delivery of anti-viral drugs to prevent vaginal HIV infection.
Bacterial lipopolysaccharide (endotoxin) is a frequent contaminant of biological specimens and is also known to be a potent inducer of β-chemokines and other soluble factors that inhibit HIV-1 infection in vitro. Though lipopolysaccharide (LPS) has been shown to stimulate the production of soluble HIV-1 inhibitors in cultures of monocyte-derived macrophages, the ability of LPS to induce similar inhibitors in other cell types is poorly characterized. Here we show that LPS exhibits potent anti-HIV activity in phytohemagglutinin-stimulated peripheral blood mononuclear cells (PBMCs) but has no detectable anti-HIV-1 activity in TZM-bl cells. The anti-HIV-1 activity of LPS in PBMCs was strongly associated with the production of β-chemokines from CD14-positive monocytes. Culture supernatants from LPS-stimulated PBMCs exhibited potent anti-HIV-1 activity when added to TZM-bl cells but, in this case, the antiviral activity appeared to be related to IFN-γ rather than to β-chemokines. These observations indicate that LPS stimulates PBMCs to produce a complex array of soluble HIV-1 inhibitors, including β-chemokines and IFN-γ, that differentially inhibit HIV-1 depending on the target cell type. The results also highlight the need to use endotoxin-free specimens to avoid artifacts when assessing HIV-1-specific neutralizing antibodies in PBMC-based assays.
Topical microbicides are self-administered, prophylactic products for protection against sexually transmitted pathogens. A large number of compounds with known anti-human immunodeficiency virus type 1 (HIV-1) inhibitory activity have been proposed as candidate topical microbicides. To identify potential leads, an in vitro screening algorithm was developed to evaluate candidate microbicides in assays that assess inhibition of cell-associated and cell-free HIV-1 transmission, entry, and fusion. The algorithm advances compounds by evaluation in a series of defined assays that generate measurements of relative antiviral potency to determine advancement or failure. Initial testing consists of a dual determination of inhibitory activity in the CD4-dependent CCR5-tropic cell-associated transmission inhibition assay and in the CD4/CCR5-mediated HIV-1 entry assay. The activity is confirmed by repeat testing, and identified actives are advanced to secondary screens to determine their effect on transmission of CXCR4-tropic viruses in the presence or absence of CD4 and their ability to inhibit CXCR4- and CCR5-tropic envelope-mediated cell-to-cell fusion. In addition, confirmed active compounds are also evaluated in the presence of human seminal plasma, in assays incorporating a pH 4 to 7 transition, and for growth inhibition of relevant strains of lactobacilli. Leads may then be advanced for specialized testing, including determinations in human cervical explants and in peripheral blood mononuclear cells against primary HIV subtypes, combination testing with other inhibitors, and additional cytotoxicity assays. PRO 2000 and SPL7013 (the active component of VivaGel), two microbicide products currently being evaluated in human clinical trials, were tested in this in vitro algorithm and were shown to be highly active against CCR5- and CXCR4-tropic HIV-1 infection.
Infection with a sexually transmitted disease (STD) increases the risk for human immunodeficiency virus (HIV) infection. Polymorphonuclear leukocytes (PMNs) are recruited into the genital tract by STD pathogens, such as Chlamydia trachomatis. Semen of HIV-infected men contains HIV associated with mononuclear cells. This study investigated the interaction among PMNs from HIV-uninfected persons, C. trachomatis, and HIV-infected cells and examined the mechanisms for enhanced HIV replication. We demonstrated that PMNs from HIV-seronegative donors induced HIV replication in mononuclear cells from 17 HIV-infected patients in medium without exogenous IL-2. HIV in the cell-free supernatants from cocultures of PMNs and patients' peripheral blood mononuclear cells (PBMCs) was replication competent, as indicated by their capacity to propagate HIV in a second round of culture using PBMCs from HIV-seronegative individuals and by the fact that proviral DNA was found in these cells. PMNs from HIV-seronegative donors increased HIV replication over 100-fold in chronically HIV-infected cell lines of the monocytic, T, and B cell lineages. Moreover, PMNs increased U1 cells' production of p24 antigen by as much as ninefold when compared with U1 cells cocultured with PBMCs. The addition of C. trachomatis to PMN and U1 coculture increased HIV replication by an additional ninefold at 24 h, whereas C. trachomatis alone had no effect on p24 antigen production by U1 cells. Thus, C. trachomatis serves not only to recruit PMNs, but also to interact with PMNs to increase HIV replication. HIV replication is triggered by contact of HIV-infected cells with PMNs, by the generation of reactive oxygen intermediates (ROIs), and by soluble factors such as TNF-alpha and IL-6. This is based on the findings that production of p24 antigen, IL-6, and TNF- alpha induced by PMNs is abrogated by disrupting or partitioning PMNs from HIV-infected cells; is inhibited by superoxide dismutase and catalase, enzymes that destroy ROIs; is enhanced by differentiated HL60 cells capable of producing ROIs; and is induced by PMNs tested negative for CMV. Furthermore, the production of ROIs is independent of HIV infection of mononuclear cells, since PMNs cocultured with HIV- uninfected parental monocytic and T cell lines generated ROIs. Therefore, the increased risk for acquiring HIV infection associated with chlamydia cervicitis may be related to the local recruitment of PMNs by C. trachomatis and the induction of infectious virus from mononuclear cells present in semen. These observations provide a rationale for strategies to reduce HIV transmission by control of STD.
The emergence of drug-resistant human immunodeficiency virus type I (HIV-1) strains presents a challenge for the design of new drugs. Anti-HIV compounds currently in use are the subject of advanced clinical trials using either HIV-1 reverse-transcriptase, viral protease, or integrase inhibitors. Recent studies show an increase in the number of HIV-1 variants resistant to anti-retroviral agents in newly infected individuals. Targeting host cell factors involved in the regulation of HIV-1 replication might be one way to combat HIV-1 resistance to the currently available anti-viral agents. A specific inhibition of HIV-1 gene expression could be expected from the development of compounds targeting host cell factors that participate in the activation of the HIV-1 LTR promoter. Here we will discuss how targeting the host can be accomplished either by using small molecules to alter the function of the host’s proteins such as p53 or cdk9, or by utilizing new advances in siRNA therapies to knock down essential host factors such as CCR5 and CXCR4. Finally, we will discuss how the viral protein interactomes should be performed to better design therapeutics against HIV-1.
We have previously shown that human defensin 5 (HD5) promotes HIV infectivity in both primary CD4+ T cells and HeLa cells expressing CD4 and CCR5. HD5 is induced in response to sexually transmitted infections (STIs) such as Chlamydia trachomatis and Neisseria gonorrhoeae, suggesting it plays a role in STI-mediated enhancement of HIV transmission. In contrast to our findings, a recent study reports that HD5 has an anti-HIV effect in primary CD4+ T cells under serum-deprived conditions. To resolve these apparently contradictory observations, we investigated experimental parameters that might contribute to contrasting effects of HD5.
Serum-deprived culture conditions were associated with anti-HIV activity. In contrast to the dependence of the HIV enhancing effect on HD5 structure, the anti-HIV activity in serum-deprived primary CD4+ T cells was independent of HD5 structure as the linear peptide [Abu] HD5 exhibited similar anti-HIV activity. Under serum deprived conditions, HD5 blocked CD4-receptor-independent HIV-1vsv infection before or after viral entry. We found that HD5 and its linear form induced significant cell death in primary CD4+ T cells under serum-deprived culture conditions. HD5-mediated apoptosis was observed as early as 2 h after addition of defensins to serum-deprived primary CD4+ T cells. In contrast to primary CD4+ T cells, HD5 did not induce cytotoxicity and promote HIV infectivity of HeLa-CD4-CCR5 cells under serum-deprived conditions.
These results indicate that under serum-deprived culture conditions HD5 is toxic for primary CD4+ T cells, warranting caution in data interpretation.
In the absence of a vaccine, there is an urgent need for the development of safe and effective topical microbicides to prevent the sexual transmission of human immunodeficiency virus type 1 (HIV-1). In this study, we proposed to develop a novel class of microbicides using syndecan as the antiviral agent. Specifically, we generated a soluble syndecan-Fc hybrid molecule by fusing the ectodomain of syndecan-1 to the Fc domain of a human IgG. We then tested the syndecan-Fc hybrid molecule for various in vitro microbicidal anti-HIV-1 properties. Remarkably, the syndecan-Fc hybrid molecule possesses multiple attractive microbicidal properties: (i) it blocks HIV-1 infection of primary targets including T cells, macrophages, and dendritic cells (DC); (ii) it exhibits a broad range of antiviral activity against primary HIV-1 isolates, multidrug resistant HIV-1 isolates, HIV-2, and simian immunodeficiency virus (SIV); (iii) it prevents transmigration of HIV-1 through human primary genital epithelial cells; (iv) it prevents HIV-1 transfer from dendritic cells to CD4+ T cells; (v) it is potent when added 2 h prior to addition of HIV-1 to target cells; (vi) it is potent at a low pH; (vii) it blocks HIV-1 infectivity when diluted in genital fluids; and (viii) it prevents herpes simplex virus infection. The heparan sulfate chains of the syndecan-Fc hybrid molecule are absolutely required for HIV-1 neutralization. Several lines of evidence suggest that the highly conserved Arg298 in the V3 region of gp120 serves as the locus for the syndecan-Fc hybrid molecule neutralization. In conclusion, this study suggests that the syndecan-Fc hybrid molecule represents the prototype of a new generation of microbicidal agents that may have promise for HIV-1 prevention.