Synthesis of bioengineered heparin chemically and biologically similar to porcine-derived products and convertible to low MW heparin.

Heparins have been invaluable therapeutic anticoagulant polysaccharides for over a century, whether used as unfractionated heparin or as low molecular weight heparin (LMWH) derivatives. However, heparin production by extraction from animal tissues presents multiple challenges, including the risk of adulteration, contamination, prion and viral impurities, limited supply, insecure supply chain, and significant batch-to-batch variability. The use of animal-derived heparin also raises ethical and religious concerns, as well as carries the risk of transmitting zoonotic diseases. Chemoenzymatic synthesis of animal-free heparin products would offer several advantages, including reliable and scalable production processes, improved purity and consistency, and the ability to produce heparin polysaccharides with molecular weight, structural, and functional properties equivalent to those of the United States Pharmacopeia (USP) heparin, currently only sourced from porcine intestinal mucosa. We report a scalable process for the production of bioengineered heparin that is biologically and compositionally similar to USP heparin. This process relies on enzymes from the heparin biosynthetic pathway, immobilized on an inert support and requires a tailored N-sulfoheparosan with N-sulfo levels similar to those of porcine heparins. We also report the conversion of our bioengineered heparin into a LMWH that is biologically and compositionally similar to USP enoxaparin. Ultimately, we demonstrate major advances to a process to provide a potential clinical and sustainable alternative to porcine-derived heparin products.

CD31, a Valuable Marker to Identify Early and Late Stages of T Cell Differentiation in the Human Thymus.

Although CD31 expression on human thymocytes has been reported, a detailed analysis of CD31 expression at various stages of T cell development in the human thymus is missing. In this study, we provide a global picture of the evolution of CD31 expression from the CD34+ hematopoietic precursor to the CD45RA+ mature CD4+ and CD8+ single-positive (SP) T cells. Using nine-color flow cytometry, we show that CD31 is highly expressed on CD34+ progenitors and stays high until the early double-positive stage (CD3-CD4+CD8α+ß-). After ß-selection, CD31 expression levels become low to undetectable. CD31 expression then increases and peaks on CD3highCD4+CD8+ double-positive thymocytes. However, following positive selection, CD31 expression differs dramatically between CD4+ and CD8+ lineages: homogeneously high on CD8 SP but lower or negative on CD4 SP cells, including a subset of CD45RA+CD31- mature CD4+ thymocytes. CD31 expression on TCRγδ thymocytes is very similar to that of CD4 SP cells. Remarkably, there is a substantial subset of semimature (CD45RA-) CD4 SP thymocytes that lack CD31 expression. Moreover, FOXP3+ and ICOS+ cells are overrepresented in this CD31- subpopulation. Despite this CD31-CD45RA- subpopulation, most egress-capable mature CD45RA+ CD4 SP thymocytes express CD31. The variations in CD31 expression appear to coincide with three major selection processes occurring during thymopoiesis: ß-selection, positive selection, and negative selection. Considering the ability of CD31 to modulate the TCR's activation threshold via the recruitment of tyrosine phosphatases, our results suggest a significant role for CD31 during T cell development.

Sphingosine-1-phosphate/sphingosine-1-phosphate receptor 1 signaling is required for migration of naive human T cells from the thymus to the periphery.

BACKGROUND: The mechanisms that govern the egress of mature thymocytes from the human thymus to the periphery remain understudied yet are of utmost importance to the field of basic immunology, as well as T-cell reconstitution in various immunodeficiencies. We examined the expression and function of sphingosine-1-phosphate (S1P) receptors in human thymocyte egress. OBJECTIVES: We aimed to determine whether S1P receptors (S1P-Rs) play a role in mature human thymocyte egress and to identify the thymocyte population or populations that express S1P-Rs and respond to S1P by migrating across a concentration gradient. METHODS: Human thymocytes were exposed to S1P in Transwell plate migration assays coupled to flow cytometry to evaluate the response to S1P of thymocytes at different stages of maturation. Constitutive S1P-R expression was quantified by means of real-time PCR in sorted thymocyte subsets and flow cytometry. S1P-R1 and Kruppel-like factor 2 expression were monitored after S1P exposure by using flow cytometry and quantitative PCR. RESULTS: S1P-R1 was the prevalent S1P receptor on mature human thymocytes (CD3(hi)CD27(+)CD69(-)), the population that also demonstrated the greatest response to S1P in migration assays. Pretreatment with FTY720, an S1P-R1 nonselective modulator significantly reduced migration and suggested a role for S1P-R2 in retaining thymocytes in the tissue. Lastly, surface S1P-R1 expression, as well S1PR1 and Kruppel-like factor 2 (KLF2) transcripts, were significantly decreased in mature thymocytes on exposure to S1P. CONCLUSION: Mature human thymocytes rely on S1P-R1 to migrate toward S1P. Taken in the context of murine work demonstrating that S1P is required for thymocyte egress to the periphery, our data highlight a new key chemokine for human thymocyte egress.

Design of monodisperse and well-defined polypeptide-based polyvalent inhibitors of anthrax toxin.

The design of polyvalent molecules, presenting multiple copies of a specific ligand, represents a promising strategy to inhibit pathogens and toxins. The ability to control independently the valency and the spacing between ligands would be valuable for elucidating structure-activity relationships and for designing potent polyvalent molecules. To that end, we designed monodisperse polypeptide-based polyvalent inhibitors of anthrax toxin in which multiple copies of an inhibitory toxin-binding peptide were separated by flexible peptide linkers. By tuning the valency and linker length, we designed polyvalent inhibitors that were over four orders of magnitude more potent than the corresponding monovalent ligands. This strategy for the rational design of monodisperse polyvalent molecules may not only be broadly applicable for the inhibition of toxins and pathogens, but also for controlling the nanoscale organization of cellular receptors to regulate signaling and the fate of stem cells.

Perhydrolase-nanotube paint composites with sporicidal and antiviral activity.

AcT (perhydrolase) containing paint composites were prepared leading to broad-spectrum decontamination. AcT was immobilized onto multi-walled carbon nanotubes (MWNTs) and then incorporated into latex-based paints to form catalytic coatings. These AcT-based paint composites showed a 6-log reduction in the viability of spores of Bacillus cereus and Bacillus anthracis (Sterne) within 60 min. The paint composites also showed >4-log reduction in the titer of influenza virus (X-31) within 10 min (initially challenged with 10(7) PFU/mL). AcT-based paint composites were also tested using various perhydrolase acyl donor substrates, including propylene glycol diacetate (PGD), glyceryl triacetate, and ethyl acetate, with PGD observed to be the best among the substrates tested for generation of peracetic acid and killing of bacillus spores. The operational stability of paint composites was also studied at different relative humidities and temperatures to simulate real-life operation.

A doxycycline-dependent human immunodeficiency virus type 1 replicates in vivo without inducing CD4+ T-cell depletion.

A novel genetic approach for the control of virus replication was used for the design of a conditionally replicating human immunodeficiency virus (HIV) variant, HIV-rtTA. HIV-rtTA gene expression and virus replication are strictly dependent on the presence of a non-toxic effector molecule, doxycycline (dox), and thus can be turned on and off at will in a graded and reversible manner. The in vivo replication capacity, pathogenicity and genetic stability of this HIV-rtTA variant were evaluated in a humanized mouse model of haematopoiesis that harbours lymphoid and myeloid components of the human immune system (HIS). Infection of dox-fed BALB Rag/γc HIS (BRG-HIS) mice with HIV-rtTA led to the establishment of a productive infection without CD4(+) T-cell depletion. The virus did not show any sign of escape from dox control for up to 10 weeks after the onset of infection. No reversion towards a functional Tat-transactivating responsive (TAR) RNA element axis was observed, confirming the genetic stability of the HIV-rtTA variant in vivo. These results demonstrate the proof of concept that HIV-rtTA replicates efficiently in vivo. HIV-rtTA is a promising tool for fundamental research to study virus-host interactions in vivo in a controlled fashion.

Light-activated nanotube-porphyrin conjugates as effective antiviral agents.

Porphyrins have been used for photodynamic therapy (PDT) against a wide range of targets like bacteria, viruses and tumor cells. In this work, we report porphyrin-conjugated multi-walled carbon nanotubes (NT-P) as potent antiviral agents. Specifically, we used Protoporphyrin IX (PPIX), which we attached to acid-functionalized multi-walled carbon nanotubes (MWNTs). We decided to use carbon nanotubes as scaffolds because of their ease of recovery from a solution through filtration. In the presence of visible light, NT-P was found to significantly reduce the ability of Influenza A virus to infect mammalian cells. NT-P may be used effectively against influenza viruses with little or no chance of them developing resistance to the treatment. Furthermore, NT-P can be easily recovered through filtration which offers a facile strategy to reuse the active porphyrin moiety to its fullest extent. Thus NT-P conjugates represent a new approach for preparing ex vivo reusable antiviral agents.

Isobutanol production by combined in vivo and in vitro metabolic engineering.

The production of the biofuel, isobutanol, in E. coli faces limitations due to alcohol toxicity, product inhibition, product recovery, and long-term industrial feasibility. Here we demonstrate an approach of combining both in vivo with in vitro metabolic engineering to produce isobutanol. The in vivo production of α-ketoisovalerate (KIV) was conducted through CRISPR mediated integration of the KIV pathway in bicistronic design (BCD) in E. coli and inhibition of competitive valine pathway using CRISPRi technology. The subsequent in vitro conversion to isobutanol was carried out with engineered enzymes for 2-ketoacid decarboxylase (KIVD) and alcohol dehydrogenase (ADH). For the in vivo production of KIV and subsequent in vitro production of isobutanol, this two-step serial approach resulted in yields of 56% and 93%, productivities of 0.62 and 0.074 g L-1 h-1, and titers of 5.6 and 1.78 g L-1, respectively. Thus, this combined biosynthetic system can be used as a modular approach for producing important metabolites, like isobutanol, without the limitations associated with in vivo production using a consolidated bioprocess.

Preparation of Low Molecular Weight Heparin from a Remodeled Bovine Intestinal Heparin.

Bovine intestinal heparins are structurally distinct from porcine intestinal heparins and exhibit lower specific anticoagulant activity (units/mg). The reduced content of N-sulfo, 3-O-sulfo glucosamine, the central and critical residue in heparin's antithrombin III binding site, is responsible for bovine intestinal heparin's reduced activity. Previous studies demonstrate that treatment of bovine intestinal heparin with 3-O-sulfotransferase in the presence of 3'-phosphoadenosine-5'-phosphosulfate afforded remodeled bovine heparin with an enhanced activity reaching the United States Pharmacopeia's requirements. Starting from this remodeled bovine intestinal heparin, we report the preparation of a bovine intestinal low molecular weight heparin having the same structural properties and anti-factor IIa and anti-factor Xa activities of Enoxaparin. Moreover, this bovine intestinal heparin-derived "Enoxaparin" showed comparable platelet factor-4 binding affinity, suggesting that it should exhibit similarly low levels of heparin induced thrombocytopeneia, HIT.

Synthesis of multivalent polymer-aptamer conjugates with enhanced inhibitory potency.

PURPOSE: We are interested in designing a modular strategy for creating potent multivalent ligands, which frequently can be used as effective inhibitors of undesired biomolecular interactions. For example, such inhibitors might prevent the self-assembly of bacterial toxins or the attachment of a virus to its host cell receptors. METHODS: We used a biocompatible polyamino acid polymer as a scaffold for grafting multiple copies of an oligonucleotide aptamer (OA). Specifically, the carboxylates on the side chains of polyglutamic acid (PGA) were modified with a thiol-reactive linker, N-aminoethyl maleimide (AEM), and thiol-functionalized OAs were attached to the maleimide moieties. The resulting conjugates were tested for their ability to compete with and inhibit the binding of unconjugated monovalent OAs to the target cell receptor. RESULTS: Multivalent PGA-OA conjugates with low, medium, and high valency were successfully prepared. The varying valency and successful purification to remove unconjugated OAs were confirmed by polyacrylamide gel electrophoresis. The resulting purified conjugates inhibited the binding of unconjugated monovalent OAs, and the measured half maximal inhibitory concentration (IC50) values corresponded to a 38-88-fold enhancement of potency on a per-aptamer basis, relative to OA alone. CONCLUSION: Multivalent conjugation of OA ligands has potential as a generally useful way to improve the potency of the interaction between the ligand and its target receptor. We have demonstrated this principle with a known OA as a proof of concept as well a synthetic strategy that can be used to synthesize multivalent conjugates of other OAs.

Nanostructured glycan architecture is important in the inhibition of influenza A virus infection.

Rapid change and zoonotic transmission to humans have enhanced the virulence of the influenza A virus (IAV). Neutralizing antibodies fail to provide lasting protection from seasonal epidemics. Furthermore, the effectiveness of anti-influenza neuraminidase inhibitors has declined because of drug resistance. Drugs that can block viral attachment and cell entry independent of antigenic evolution or drug resistance might address these problems. We show that multivalent 6'-sialyllactose-polyamidoamine (6SL-PAMAM) conjugates, when designed to have well-defined ligand valencies and spacings, can effectively inhibit IAV infection. Generation 4 (G4) 6SL-PAMAM conjugates with a spacing of around 3 nm between 6SL ligands (S3-G4) showed the strongest binding to a hemagglutinin trimer (dissociation constant of 1.6 × 10-7 M) and afforded the best inhibition of H1N1 infection. S3-G4 conjugates were resistant to hydrolysis by H1N1 neuraminidase. These conjugates protected 75% of mice from a lethal challenge with H1N1 and prevented weight loss in infected animals. The structure-based design of multivalent nanomaterials, involving modulation of nanoscale backbone structures and number and spacing between ligands, resulted in optimal inhibition of IAV infection. This approach may be broadly applicable for designing effective and enduring therapeutic protection against human or avian influenza viruses.

APOBEC3G ubiquitination by Nedd4-1 favors its packaging into HIV-1 particles.

APOBEC3G is a cytidine deaminase that limits the replication of many retroviruses. This antiviral host factor is packaged into retrovirus particles, where it targets single-stranded DNA generated during reverse transcription and induces up to 2% of G-to-A mutations, which are lethal for the HIV-1 provirus. Vif protein counteracts this antiviral factor by decreasing its packaging into lentivirus particles. Here, we demonstrate that Nedd4-1, an HECT E3 ubiquitin ligase, interacts with APOBEC3G, through its WW2 and WW3 domains. As a result of this interaction, APOBEC3G undergoes post-translational modification by addition of ubiquitin moieties. Accordingly, we demonstrate that the dominant negative Nedd4-1 C/S form prevents APOBEC3G ubiquitination. Moreover, the packaging of APOBEC3G into Pr55 Gag virus-like particles and into HIV-1 virions is reduced when Nedd4-1 C/S is expressed. During HIV-1 viral production in the presence of APOBEC3G, Nedd4-1 C/S restores partially the infectivity of Deltavif HIV-1. We conclude that the ubiquitination of APOBEC3G by Nedd4-1 favors its targeting to the virus assembly site where APOBEC3G interacts with Gag and is packaged into HIV-1 particles in the absence of Vif.

The tyrosine kinases Fyn and Hck favor the recruitment of tyrosine-phosphorylated APOBEC3G into vif-defective HIV-1 particles.

The main function of Vif is to limit the antiviral activity of APOBEC3G by counteracting its packaging into HIV-1 virions. In this work, we examine the possible functional interactions between Vif, APOBEC3G, and two Src family tyrosine kinases, Fyn and Hck, present in T lymphocytes and in monocyte-macrophages, respectively. By GST pull-down, we show that the SH3 domains of Fyn and Hck, and the corresponding full-length proteins bind Vif of HIV-1. One consequence of this interaction is a reduction in their catalytic activity. Interestingly, we also observed that APOBEC3G can be phosphorylated on tyrosine in the presence of Fyn or Hck, suggesting that both kinases may regulate APOBEC3G function. Accordingly, we demonstrate that in the presence of Fyn or Hck and in the absence of Vif, the overall level of APOBEC3G incorporated into HIV-1 particles is decreased, whereas the level of encapsidation of its phosphorylated form is significantly enhanced.

HIV-1 and MLV Gag proteins are sufficient to recruit APOBEC3G into virus-like particles.

The cytidine deaminase hAPOBEC3G is an antiviral human factor that counteracts the replication of HIV-1 in absence of the Vif protein. hAPOBEC3G is packaged into virus particles and lethally hypermutates HIV-1. In this work, we examine the mechanisms governing hAPOBEC3G packaging. By GST pull-down and co-immunoprecipitation assays, we show that hAPOBEC3G binds to HIV-1 Pr55 Gag and its NC domain and to the RT and IN domains contained in Pr160 Gag-Pol. We demonstrate that the expression of HIV-1 Gag is sufficient to induce the packaging of hAPOBEC3G into Gag particles. Gag-Pol polypeptides containing RT and IN domains, as well as HIV-1 genomic RNA, seem not to be necessary for hAPOBEC3G packaging. Lastly, we show that hAPOBEC3G and its murine ortholog are packaged into HIV-1 and MLV Gag particles. We conclude that the Gag polypeptides from distant retroviruses have conserved domains allowing the packaging of the host antiviral factor APOBEC3G.

The Vif protein of human immunodeficiency virus type 1 is posttranslationally modified by ubiquitin.

The viral infectivity factor (Vif), one of the six HIV-1 auxiliary genes, is absolutely necessary for productive infection in primary CD4-positive T lymphocytes and macrophages. Vif overcomes the antiviral function of the host factor APOBEC3G. To better understand this mechanism, it is of interest to characterize cellular proteins that interact with Vif and may regulate its function. Here, we show that Vif binds to hNedd4 and AIP4, two HECT E3 ubiquitin ligases. WW domains present in those HECT enzymes contribute to the binding of Vif. Moreover, the region of Vif, which includes amino acids 20-128 and interacts with the hNedd4 WW domains, does not contain proline-rich stretches. Lastly, we show that Vif undergoes post-translational modifications by addition of ubiquitin both in cells overexpressing Vif and in cells expressing HIV-1 provirus. Vif is mainly mono-ubiquitinated, a modification known to address the Gag precursor to the virus budding site.