TZM-bl Reporter Cells Research Articles

The TZM-bl Reporter Cell Line Expresses Kynureninase That Can Neutralize 2F5-like Antibodies in the HIV-1 Neutralization Assay.

Induction of broadly neutralizing antibodies targeting ectodomain of the transmembrane (TM) glycoprotein gp41 HIV-1 provides a basis for the development of a universal anti-viral vaccine. The HeLa cell-derived TZM-bl reporter cell line is widely used for the estimation of lentiviruses neutralization by immune sera. The cell line is highly permissive to infection by most strains of HIV, SIV, and SHIV. Here we demonstrated that TZM-bl cells express a 48 kDa non-glycosylated protein (p48) recognized by broadly neutralizing monoclonal antibody (mAb) 2F5 targeting the ELDKWA (aa 669–674) epitope of gp41TM of HIV-1. A significant amount of p48 was found in the cell supernatant. The protein was identified as human kynureninase (KYNU), which has the ELDKWA epitope. The protein is further called “p48 KYNU”. The HIV-1 neutralization by mAb 2F5 and 4E10 in the presence of p48KYNU was tested on Jurkat and TZM-bl cells. It was demonstrated that p48KYNU reduces neutralization by 2F5-like antibodies, but it has almost no effect on mAb 4E10. Therefore, p48KYNU can attenuate HIV-1 neutralization by 2F5-like antibodies and hence create false-negative results. Thus, previously tested immune sera that recognized the ELDKWA-epitope and demonstrated a “weak neutralization” of HIV-1 in TZM-bl assay should be reevaluated.

A Novel High Throughput, Parallel Infection Assay for Determining the Replication Capacities of 346 Primary HIV-1 Isolates of the Zurich Primary HIV-1 Infection Study in Primary Cells.

HIV-1 replication capacity is an important characteristic to understand the replication competence of single variants or virus populations. It can further aid in the understanding of HIV-1 pathogenicity, disease progression, and drug resistance mutations. To effectively study RC, many assays have been established. However, there is still demand for a high throughput replication capacity assay using primary cells which is robust and reproducible. In this study, we established such an assay and validated it using 346 primary HIV-1 isolates from patients enrolled in the Zurich Primary HIV Infection study (ZPHI) and two control viruses, HIV-1 JR-CSFWT and HIV-1 JR-CSFK65R_M184V. Replication capacity was determined by measuring the viral growth on PBMCs over 10 days by longitudinally transferring cell culture supernatant to TZM-bl reporter cells. By utilizing the TZM-bl luciferase reporter assay, we determined replication capacity by measuring viral infectivity. The simplicity of the experimental setup allowed for all 346 primary HIV-1 isolates to be replicated at one time. Although the infectious input dose for each virus was normalized, a broad range of replication capacity values over 4 logs was observed. The approach was confirmed by two repeated experiments and we demonstrated that the reproducibility of the replication capacity values is statistically comparable between the two separate experiments. In summary, these results endorse our high throughput replication capacity assay as reproducible and robust and can be utilized for large scale HIV-1 replication capacity experiments in primary cells.

Nef Dimerization is Required for Antagonism of the Host Cell Restriction Factor, SERINC5

HIV‐1 Nef is a virulence factor that is essential for immune escape of HIV‐infected cells, viral pathogenesis and AIDS progression. Nef lacks intrinsic biochemical activity, functioning instead by interacting with host cell effector proteins to hijack multiple signaling and endocytic pathways. Many Nef functions depend on dimerization of the protein, including enhancement of viral infectivity, replication, and immune cell receptor downregulation. Work in our lab, through both X‐ray crystallography and virological assays, has shown that distinct hydrophobic residues (L112, Y115, F121) are key in Nef dimer formation, and disruption of these residues significantly impairs many Nef functions. Pharmacologic disruption of the Nef homodimer may therefore represent a novel approach to antiretroviral therapy.Here we investigated whether Nef dimerization is required for antagonism of the host cell restriction factor SERINC5 (S5). S5 is a potent suppressor of HIV‐1 infectivity that is antagonized by Nef via binding and downregulating S5 from the surface of infected cells, preventing its incorporation into nascent virions. For our studies, HIV‐1 virions were produced in 293T cells transfected with the HIV‐1 provirus and S5, Jurkat WT and S5‐knockout cells, or donor PBMCs (which naturally express high levels of S5). Viral supernatants were harvested from these “producer” cells and normalized for p24 AlphaLISA. Infectivity of producer‐cell viruses was assessed using TZM‐bl reporter cells, which couple the HIV‐1 LTR to expression of luciferase. Mutation of conserved Nef residues essential for homodimerization (L112A/D, Y115D, F121A) reduced the infectivity of HIV‐1 produced from all three producer cell lines in a S5‐dependent manner. S5 incorporation into virions was increased in the presence of Nef dimer interface mutants, suggesting that dimerization is important in its ability to exclude S5 from newly synthesized virions. Recruitment, downregulation, and endocytic trafficking of S5 by wild type and dimerization‐defective Nef mutants were also examined via confocal microscopy. Nef dimer interface mutants also failed to induce colocalization of S5 with Rab7+ late endosomes, suggesting that Nef dimerization is essential for S5 internalization and degradation via the lyso‐endosomal pathway. By contrast, Nef mutants retained their ability to interact with both S5 and subunits of the AP‐2 clathrin adaptor protein essential for S5 endocytosis, as shown via bimolecular fluorescence complementation. Taken together, these results demonstrate that downregulation of S5 and subsequent enhancement of viral infectivity requires Nef homodimerization, providing further support for pharmacological targeting of Nef quaternary structure in HIV‐1 cure strategies.

Tight-Binding Hydroxypyrazole HIV-1 Nef Inhibitors Suppress Viral Replication in Donor Mononuclear Cells and Reverse Nef-Mediated MHC-I Downregulation.

The HIV-1 Nef accessory factor is critical to the viral life cycle in vivo and promotes immune escape of infected cells via downregulation of cell-surface MHC-I. Previously, we discovered small molecules that bind directly to Nef and block many of its functions, including enhancement of viral infectivity and replication in T cell lines. These compounds also restore cell-surface MHC-I expression in HIV-infected CD4 T cells from AIDS patients, enabling recognition and killing by autologous cytotoxic T lymphocytes (CTLs). In this study, we describe the synthesis and evaluation of a diverse set of analogs based on the original hydroxypyrazole Nef inhibitor core. All analogs were screened for the interaction with recombinant HIV-1 Nef by surface plasmon resonance (SPR) and for antiretroviral activity in TZM-bl reporter cells infected with HIV-1. Active analogs were ranked on the basis of an activity score that integrates three aspects of the SPR data (affinity, residence time, and extent of binding) with antiretroviral activity. The top scoring compounds bound tightly to Nef by SPR, with KD values in the low nM to pM range, and displayed very slow dissociation from their Nef target. These analogs also suppressed HIV-1 replication in donor peripheral blood mononuclear cells (PBMCs) with IC50 values in the 1-10 nM range without cytotoxicity, inhibited Nef-mediated IL-2-inducible tyrosine kinase (Itk) and hematopoietic cell kinase (Hck) activation, and rescued MHC-I downregulation in a Nef-transfected T cell line. The development of Nef inhibitors based on the structure-activity relationships defined here has promise as a new approach to antiretroviral therapy that includes a path to eradication of HIV-infected cells via the adaptive immune response.

Efficient inactivation of pseudotyped HIV-based lentiviral vectors and infectious HIV

Lentiviral vectors and lentiviruses are important tools for basic and applied biomedical research. Yet, biosafety regulations from legal authorities have to be fulfilled when transferring BSL-2 to -3 vectors/viruses to facilities with lower biosafety level. Here, we (re-)evaluated different chemical and thermal approaches to inactivate vesicular stomatitis virus G-protein (VSV-G) pseudotyped lentiviral vectors and either wildtype or VSV-G pseudotyped human immunodeficiency viruses (HIV). Aldehydes, detergents and alcohols were as effective as thermal inactivation procedures to efficiently inactivate purified lentiviral vectors and replication-competent HIV. In addition, no residual infectivity was detected when inactivating HIV-infected TZM-bl reporter cells with selected detergents and aldehydes. Thus, our established inactivation protocols can be used by other laboratories working with lentiviral vectors or infectious lentiviruses and provide a template for viruses with similar physicochemical properties.

Hydrophobic-core PEGylated graft copolymer-stabilized nanoparticles composed of insoluble non-nucleoside reverse transcriptase inhibitors exhibit strong anti-HIV activity

Benzophenone-uracil (BPU) scaffold-derived candidate compounds are efficient non-nucleoside reverse transcriptase inhibitors (NNRTI) with extremely low solubility in water. We proposed to use hydrophobic core (methoxypolyethylene glycol-polylysine) graft copolymer (HC-PGC) technology for stabilizing nanoparticle-based formulations of BPU NNRTI in water. Co-lyophilization of NNRTI/HC-PGC mixtures resulted in dry powders that could be easily reconstituted with the formation of 150–250 nm stable nanoparticles (NP). The NP and HC-PGC were non-toxic in experiments with TZM-bl reporter cells. Nanoparticles containing selected efficient candidate Z107 NNRTI preserved the ability to inhibit HIV-1 reverse transcriptase polymerase activities with no appreciable change of EC50. The formulation with HC-PGC bearing residues of oleic acid resulted in nanoparticles that were nearly identical in anti-HIV-1 potency when compared to Z107 solutions in DMSO (EC50=7.5±3.8 vs. 8.2±5.1 nM). Therefore, hydrophobic core macromolecular stabilizers form nanoparticles with insoluble NNRTI while preserving the antiviral activity of the drug cargo.

Antiviral activity of cystatin C against HIV.

Antiviral activity of cystatin C against HIV.

Innate immunity in the vagina (Part II): Anti-HIV activity and antiviral content of human vaginal secretions.

Whether the concentrations of antiviral proteins, and anti-HIV activity, within human vaginal secretions change across the menstrual cycle is unknown. Using a menstrual cup, vaginal secretions from pre-menopausal women were recovered at the proliferative (d6-8), mid-cycle (d13-15), and secretory (d21-23) stages of the menstrual cycle. Antiviral protein concentration was determined by ELISA, and anti-HIV activity assessed using the TZM-bl reporter cell line. CCL20, RANTES, elafin, HBD2, SDF-1α, and IL-8 levels were detectable in the secretions. Vaginal secretions had anti-HIV activity against specific clade B strains of HIV, with significant inhibition of IIIB and increased infectivity of transmitted/founder CH077.t. No significant differences in either antiviral protein concentration or anti-HIV activity with respect to menstrual cycle stage were measured, but marked differences were observed in both parameters over the course of the cycle between different women and in consecutive cycles from the same woman. The vagina contains a complement of antiviral proteins. The variation in anti-HIV activity demonstrates that immune protection in the vagina is not constant. Intra- and interindividual variations suggest that factors in addition to sex hormones influence antiviral protection. Lastly, the menstrual cup is a new model for recovering undiluted vaginal secretions from women throughout their reproductive life.

Vaginal concentrations of lactic acid potently inactivate HIV

ObjectivesWhen Lactobacillus spp. dominate the vaginal microbiota of women of reproductive age they acidify the vagina to pH <4.0 by producing ∼1% lactic acid in a nearly racemic mixture of d- and l-isomers. We determined the HIV virucidal activity of racemic lactic acid, and its d- and l-isomers, compared with acetic acid and acidity alone (by the addition of HCl).MethodsHIV-1 and HIV-2 were transiently treated with acids in the absence or presence of human genital secretions at 37°C for different time intervals, then immediately neutralized and residual infectivity determined in the TZM-bl reporter cell line.Resultsl-lactic acid at 0.3% (w/w) was 17-fold more potent than d-lactic acid in inactivating HIVBa-L. Complete inactivation of different HIV-1 subtypes and HIV-2 was achieved with ≥0.4% (w/w) l-lactic acid. At a typical vaginal pH of 3.8, l-lactic acid at 1% (w/w) more potently and rapidly inactivated HIVBa-L and HIV-1 transmitter/founder strains compared with 1% (w/w) acetic acid and with acidity alone, all adjusted to pH 3.8. A final concentration of 1% (w/w) l-lactic acid maximally inactivated HIVBa-L in the presence of cervicovaginal secretions and seminal plasma. The anti-HIV activity of l-lactic acid was pH dependent, being abrogated at neutral pH, indicating that its virucidal activity is mediated by protonated lactic acid and not the lactate anion.Conclusionsl-lactic acid at physiological concentrations demonstrates potent HIV virucidal activity distinct from acidity alone and greater than acetic acid, suggesting a protective role in the sexual transmission of HIV.

Cytolytic Nanoparticles Attenuate HIV-1 Infectivity

We investigated whether cytolytic melittin peptides could inhibit HIV-1 infectivity when carried in a nanoparticle construct that might be used as a topical vaginal virucide. Free melittin and melittin-loaded nanoparticles were prepared and compared for cytotoxicity and their ability to inhibit infectivity by CXCR4 and CCR5 tropic HIV-1 strains. TZM-bl reporter cells expressing luciferase under the control of the HIV-1 promoter were incubated with HIV-1 NLHX (CXCR4) or HIV-1 NLYU2 (CCR5) viral strains and different doses of soluble CD4 (positive control) or free melittin to determine infectivity and viability. Melittin-loaded nanoparticles were formulated and different doses tested against VK2 vaginal epithelial cells to determine cell viability. Based on VK2 viability, melittin nanoparticles were tested for prevention of CXCR4 and CCR5 tropic HIV-1 infectivity and viability of TZM-bl reporter cells. Low-speed centrifugation was used to compare the ability of blank non-melittin nanoparticles and melittin nanoparticles to capture CCR5 tropic HIV-1. As expected, the soluble CD4 positive control inhibited CXCR4 (50% inhibitory concentration [IC₅₀] 3.7 μg/ml) and CCR5 (IC₅₀ 0.03 μg/ml) tropic HIV-1 infectivity. Free melittin doses <2 μM were not cytotoxic and were highly effective in reducing HIV-1 infectivity for both CXCR4 and CCR5 strains in TZM-bl reporter cells, while VK2 vaginal cell viability was adversely affected at all free melittin doses tested. However, VK2 cell viability was not affected at any dose of melittin-loaded nanoparticles. Melittin nanoparticles safely and significantly decreased CXCR4 (IC₅₀ 2.4 μM and IC₉₀ 6.9 μM) and CCR5 (IC₅₀ 3.6 μM and IC₉₀ 11.4 μM) strain infectivity of TZM-bl reporter cells. Furthermore, melittin nanoparticles captured more HIV-1 than blank nanoparticles. These data illustrate the first proof-of-concept for therapeutic and safe nanoparticle-mediated inhibition of HIV-1 infectivity. Future investigations appear warranted to explore the antiviral prophylactic potential of melittin nanoparticles to capture, disrupt and prevent initial infection with HIV-1 or potentially other enveloped viruses.

Impact of sustained RNAi-mediated suppression of cellular cofactor Tat-SF1 on HIV-1 replication in CD4+ T cells

BackgroundConventional anti-HIV drug regimens targeting viral enzymes are plagued by the emergence of drug resistance. There is interest in targeting HIV-dependency factors (HDFs), host proteins that the virus requires for replication, as drugs targeting their function may prove protective. Reporter cell lines provide a rapid and convenient method of identifying putative HDFs, but this approach may lead to misleading results and a failure to detect subtle detrimental effects on cells that result from HDF suppression. Thus, alternative methods for HDF validation are required. Cellular Tat-SF1 has long been ascribed a cofactor role in Tat-dependent transactivation of viral transcription elongation. Here we employ sustained RNAi-mediated suppression of Tat-SF1 to validate its requirement for HIV-1 replication in a CD4+ T cell-derived line and its potential as a therapeutic target.ResultsshRNA-mediated suppression of Tat-SF1 reduced HIV-1 replication and infectious particle production from TZM-bl reporter cells. This effect was not a result of increased apoptosis, loss of cell viability or an immune response. To validate its requirement for HIV-1 replication in a more relevant cell line, CD4+ SupT1 cell populations were generated that stably expressed shRNAs. HIV-1 replication was significantly reduced for two weeks (~65%) in cells with depleted Tat-SF1, although the inhibition of viral replication was moderate when compared to SupT1 cells expressing a shRNA targeting the integration cofactor LEDGF/p75. Tat-SF1 suppression was attenuated over time, resulting from decreased shRNA guide strand expression, suggesting that there is a selective pressure to restore Tat-SF1 levels.ConclusionsThis study validates Tat-SF1 as an HDF in CD4+ T cell-derived SupT1 cells. However, our findings also suggest that Tat-SF1 is not a critical cofactor required for virus replication and its suppression may affect cell growth. Therefore, this study demonstrates the importance of examining HIV-1 replication kinetics and cytotoxicity in cells with sustained HDF suppression to validate their therapeutic potential as targets.

Milk Matters: Soluble Toll-Like Receptor 2 (sTLR2) in Breast Milk Significantly Inhibits HIV-1 Infection and Inflammation

The majority of infants who breastfeed from their HIV-positive mothers remain uninfected despite constant and repeated exposure to virus over weeks to years. This phenomenon is not fully understood but has been closely linked to innate factors in breast milk (BM). Most recently we have focused on one such innate factor, soluble Toll-like receptor 2 (sTLR2) for its significant contribution as an inhibitor of inflammation triggered by bacterial and viral antigens. We hypothesized that sTLR2 in BM inhibits immune activation/inflammation and HIV-1 infection. sTLR2 protein profiles were analyzed in HIV-uninfected BM and showed dramatic variability in expression concentration and predominant sTLR2 forms between women. sTLR2 immunodepleted BM, versus mock-depleted BM, incubated with Pam3CSK4 lead to significant increases in IL-8 production in a TLR2-dependant fashion in U937, HEK293-TLR2, and Caco-2. Importantly, TLR2-specific polyclonal and monoclonal antibody addition to BM prior to cell-free R5 HIV-1 addition led to significantly (P<0.01, P<0.001, respectively) increased HIV-1 infection in TZM-bl reporter cells. To confirm these findings, sTLR2-depletion in BM led to significantly (P<0.001) increased HIV-1 infection in TZM-bl cells. Notably, immunodepletion does not allow for the complete removal of sTLR2 from BM, thus functional testing shown here may underestimate the total effect elicited by sTLR2 against HIV-1 and synthetic bacterial ligand. This study provides evidence for the first time that sTLR2 in BM may provide a dual protective role for infants breastfeeding from their HIV-infected mothers by; (1) immunomodulating pro-inflammatory responses to bacterial ligands, and (2) directly inhibiting cell-free HIV-1 infection. Thus, sTLR2 in BM may be critical to infant health and prove beneficial in decreasing vertical HIV-1 transmission to infants.

Assessment of the range of the HIV-1 infectivity enhancing effect of individual human semen specimen and the range of inhibition by EGCG

Recently, it has been shown that human ejaculate enhances human immunodeficiency virus 1 (HIV-1) infectivity. Enhancement of infectivity is conceived to be mediated by amyloid filaments from peptides that are proteolytically released from prostatic acid phosphatase (PAP), termed Semen-derived Enhancer of Virus Infection (SEVI). The aim of this study was to test the range of HIV-1 infectivity enhancing properties of a large number of individual semen samples (n = 47) in a TZM-bl reporter cell HIV infection system. We find that semen overall increased infectivity to 156% of the control experiment without semen, albeit with great inter- and intraindividual variability (range -53%-363%). Using transmission electron microscopy, we provide evidence for SEVI fibrils in fresh human semen for the first time. Moreover, we confirm that the infectivity enhancing property can be inhibited by the major green tea ingredient epigallocatechin-3-gallate (EGCG) at non-toxic concentrations. The median inhibition of infection by treatment with 0.4 mM EGCG was 70.6% (p < 0.0001) in our cohort. Yet, there were substantial variations of inhibition and in a minority of samples, infectivity enhancement was not inhibited by EGCG treatment at all. Thus, topical application of EGCG may be a feasible additional measure to prevent the sexual transmission of HIV. However, the reasons for the variability in the efficacy of the abrogation of semen-mediated enhancement of HIV-1 infectivity and EGCG efficacy have to be elucidated before therapeutic trials can be conducted.

Proviral HIV-genome-wide and pol-gene specific zinc finger nucleases: usability for targeted HIV gene therapy.

BackgroundInfection with HIV, which culminates in the establishment of a latent proviral reservoir, presents formidable challenges for ultimate cure. Building on the hypothesis that ex-vivo or even in-vivo abolition or disruption of HIV-gene/genome-action by target mutagenesis or excision can irreversibly abrogate HIV's innate fitness to replicate and survive, we previously identified the isoschizomeric bacteria restriction enzymes (REases) AcsI and ApoI as potent cleavers of the HIV-pol gene (11 and 9 times in HIV-1 and 2, respectively). However, both enzymes, along with others found to cleave across the entire HIV-1 genome, slice (SX) at palindromic sequences that are prevalent within the human genome and thereby pose the risk of host genome toxicity. A long-term goal in the field of R-M enzymatic therapeutics has thus been to generate synthetic restriction endonucleases with longer recognition sites limited in specificity to HIV. We aimed (i) to assemble and construct zinc finger arrays and nucleases (ZFN) with either proviral-HIV-pol gene or proviral-HIV-1 whole-genome specificity respectively, and (ii) to advance a model for pre-clinically testing lentiviral vectors (LV) that deliver and transduce either ZFN genotype.Methods and ResultsFirst, we computationally generated the consensus sequences of (a) 114 dsDNA-binding zinc finger (Zif) arrays (ZFAs or ZifHIV-pol) and (b) two zinc-finger nucleases (ZFNs) which, unlike the AcsI and ApoI homeodomains, possess specificity to >18 base-pair sequences uniquely present within the HIV-pol gene (ZifHIV-polFN). Another 15 ZFNs targeting >18 bp sequences within the complete HIV-1 proviral genome were constructed (ZifHIV-1FN). Second, a model for constructing lentiviral vectors (LVs) that deliver and transduce a diploid copy of either ZifHIV-polFN or ZifHIV-1FN chimeric genes (termed LV- 2xZifHIV-polFN and LV- 2xZifHIV-1FN, respectively) is proposed. Third, two preclinical models for controlled testing of the safety and efficacy of either of these LVs are described using active HIV-infected TZM-bl reporter cells (HeLa-derived JC53-BL cells) and latent HIV-infected cell lines.ConclusionLV-2xZifHIV-polFN and LV- 2xZifHIV-1FN may offer the ex-vivo or even in-vivo experimental opportunity to halt HIV replication functionally by directly abrogating HIV-pol-gene-action or disrupting/excising over 80% of the proviral HIV DNA from latently infected cells.

Poor Performance of Bioinformatics Programs for Genotypic Prediction of Coreceptor Usage of HIV-1 Group O Isolates

Poor Performance of Bioinformatics Programs for Genotypic Prediction of Coreceptor Usage of HIV-1 Group O Isolates

Recent advances in the characterization of HIV-1 neutralization assays for standardized evaluation of the antibody response to infection and vaccination

In AIDS vaccine development the pendulum has swung towards a renewed emphasis on the potential role for neutralizing antibodies in a successful global vaccine. It is recognized that vaccine-induced antibody performance, as assessed in the available neutralization assays, may well serve as a “gatekeeper” for HIV-1 subunit vaccine prioritization and advancement. As a result, development of a standardized platform for reproducible measurement of neutralizing antibodies has received considerable attention. Here we review current advancements in our knowledge of the performance of different types of antibodies in a traditional primary cell neutralization assay and the newer, more standardized TZM-bl reporter cell line assay. In light of recently revealed differences (see accompanying article) in the results obtained in these two neutralization formats, parallel evaluation with both platforms should be contemplated as an interim solution until a better understanding of immune correlates of protection is achieved.