ENPP1 enzyme replacement therapy improves blood pressure and cardiovascular function in a mouse model of generalized arterial calcification of infancy.

Generalized arterial calcification of infancy (GACI) is a rare, life-threatening disorder caused by loss-of-function mutations in the gene encoding ectonucleotide pyrophosphatase phosphodiesterase 1 (), which normally hydrolyzes extracellular ATP into AMP and pyrophosphate (PP). The disease is characterized by extensive arterial calcification and stenosis of large- and medium-sized vessels, leading to vascular-related complications of hypertension and heart failure. There is currently no effective treatment available, but bisphosphonates - nonhydrolyzable PP analogs - are being used off-label to reduce arterial calcification, although this has no reported impact on the hypertension and cardiac dysfunction features of GACI.

Design and preclinical characterization of ALXN1210: A novel anti-C5 antibody with extended duration of action.

Eculizumab, a monoclonal antibody (mAb) directed against complement protein C5, is considered to be the current standard of care for patients with paroxysmal nocturnal hemoglobinuria (PNH) and atypical hemolytic uremic syndrome. This study describes the generation and preclinical attributes of ALXN1210, a new long-acting anti-C5 mAb, obtained through select modifications to eculizumab to both largely abolish target-mediated drug disposition (TMDD) and increase recycling efficiency via the neonatal Fc receptor (FcRn). To attenuate the effect of TMDD on plasma terminal half-life (t1/2), histidine substitutions were engineered into the complementarity-determining regions of eculizumab to enhance the dissociation rate of the mAb:C5 complex in the acidic early endosome relative to the slightly basic pH of blood.

Efficacy of Targeted Complement Inhibition in Experimental C3 Glomerulopathy.

C3 glomerulopathy refers to renal disorders characterized by abnormal accumulation of C3 within the kidney, commonly along the glomerular basement membrane (GBM). C3 glomerulopathy is associated with complement alternative pathway dysregulation, which includes functional defects in complement regulator factor H (FH). There is no effective treatment for C3 glomerulopathy.

Stable 293 T and CHO cell lines expressing cleaved, stable HIV-1 envelope glycoprotein trimers for structural and vaccine studies.

Background: Recombinant soluble, cleaved HIV-1 envelope glycoprotein SOSIP.664 gp140 trimers based on the subtype A BG505 sequence are being studied structurally and tested as immunogens in animals. For these trimers to become a vaccine candidate for human trials, they would need to be made in appropriate amounts at an acceptable quality.

Influences on trimerization and aggregation of soluble, cleaved HIV-1 SOSIP envelope glycoprotein.

We describe methods to improve the properties of soluble, cleaved gp140 trimers of the human immunodeficiency virus type 1 (HIV-1) envelope glycoproteins (Env) for use in structural studies and as immunogens. In the absence of nonionic detergents, gp140 of the KNH1144 genotype, terminating at residue 681 in gp41 (SOSIP.681), has a tendency to form higher-order complexes or aggregates, which is particularly undesirable for structure-based research.

Supersite of immune vulnerability on the glycosylated face of HIV-1 envelope glycoprotein gp120.

A substantial proportion of the broadly neutralizing antibodies (bnAbs) identified in certain HIV-infected donors recognize glycan-dependent epitopes on HIV-1 gp120. Here we elucidate how the bnAb PGT 135 binds its Asn332 glycan-dependent epitope from its 3.1-Å crystal structure with gp120, CD4 and Fab 17b.

Broadly neutralizing antibody PGT121 allosterically modulates CD4 binding via recognition of the HIV-1 gp120 V3 base and multiple surrounding glycans.

New broad and potent neutralizing HIV-1 antibodies have recently been described that are largely dependent on the gp120 N332 glycan for Env recognition. Members of the PGT121 family of antibodies, isolated from an African donor, neutralize ∼70% of circulating isolates with a median IC50 less than 0.05 µg ml(-1).

Multifaceted mechanisms of HIV inhibition and resistance to CCR5 inhibitors PSC-RANTES and Maraviroc.

Small-molecule CCR5 antagonists, such as maraviroc (MVC), likely block HIV-1 through an allosteric, noncompetitive inhibition mechanism, whereas inhibition by agonists such as PSC-RANTES is less defined and may involve receptor removal by cell surface downregulation, competitive inhibition by occluding the HIV-1 envelope binding, and/or allosteric effects by altering CCR5 conformation. We explored the inhibitory mechanisms of maraviroc and PSC-RANTES by employing pairs of virus clones with differential sensitivities to these inhibitors. Intrinsic PSC-RANTES-resistant virus (YA versus RT) or those selected in PSC-RANTES treated macaques (M584 versus P3-4) only displayed resistance in multiple-cycle assays or with a CCR5 mutant that cannot be downregulated.

Asymmetric recognition of the HIV-1 trimer by broadly neutralizing antibody PG9.

PG9 is the founder member of an expanding family of glycan-dependent human antibodies that preferentially bind the HIV (HIV-1) envelope (Env) glycoprotein (gp) trimer and broadly neutralize the virus. Here, we show that a soluble SOSIP.664 gp140 trimer constructed from the Clade A BG505 sequence binds PG9 with high affinity (∼11 nM), enabling structural and biophysical characterizations of the PG9:Env trimer complex.

Succession in the gut microbiome following antibiotic and antibody therapies for Clostridium difficile.

Antibiotic disruption of the intestinal microbiota may cause susceptibility to pathogens that is resolved by progressive bacterial outgrowth and colonization. Succession is central to ecological theory but not widely documented in studies of the vertebrate microbiome. Here, we study succession in the hamster gut after treatment with antibiotics and exposure to Clostridium difficile.

Protection against Clostridium difficile infection with broadly neutralizing antitoxin monoclonal antibodies.

The spore-forming bacterium Clostridium difficile represents the principal cause of hospital-acquired diarrhea and pseudomembranous colitis worldwide. C. difficile infection (CDI) is mediated by 2 bacterial toxins, A and B; neutralizing these toxins with monoclonal antibodies (mAbs) provides a potential nonantibiotic strategy for combating the rising prevalence, severity, and recurrence of CDI.

Partial enzymatic deglycosylation preserves the structure of cleaved recombinant HIV-1 envelope glycoprotein trimers.

The trimeric envelope glycoprotein complex (Env) is the focus of vaccine development programs aimed at generating protective humoral responses to human immunodeficiency virus type 1 (HIV-1). N-Linked glycans, which constitute almost half of the molecular mass of the external Env domains, produce considerable structural heterogeneity and are a major impediment to crystallization studies. Moreover, by shielding the peptide backbone, glycans can block attempts to generate neutralizing antibodies against a substantial subset of potential epitopes when Env proteins are used as immunogens.

A potent and broad neutralizing antibody recognizes and penetrates the HIV glycan shield.

The HIV envelope (Env) protein gp120 is protected from antibody recognition by a dense glycan shield. However, several of the recently identified PGT broadly neutralizing antibodies appear to interact directly with the HIV glycan coat. Crystal structures of antigen-binding fragments (Fabs) PGT 127 and 128 with Man(9) at 1.

Trimeric HIV-1 glycoprotein gp140 immunogens and native HIV-1 envelope glycoproteins display the same closed and open quaternary molecular architectures.

The initial step in HIV-1 infection occurs with the binding of cell surface CD4 to trimeric HIV-1 envelope glycoproteins (Env), a heterodimer of a transmembrane glycoprotein (gp41) and a surface glycoprotein (gp120). The design of soluble versions of trimeric Env that display structural and functional properties similar to those observed on intact viruses is highly desirable from the viewpoint of designing immunogens that could be effective as vaccines against HIV/AIDS. Using cryoelectron tomography combined with subvolume averaging, we have analyzed the structure of SOSIP gp140 trimers, which are cleaved, solubilized versions of the ectodomain of trimeric HIV-1 Env.

Phase 2a study of the CCR5 monoclonal antibody PRO 140 administered intravenously to HIV-infected adults.

The anti-CCR5 antibody PRO 140 has shown potent and prolonged antiretroviral activity in subjects infected with CCR5-tropic (R5) HIV-1. Prior studies have examined single intravenous doses ranging up to 5 mg/kg of body weight or up to three subcutaneous doses ranging up to 324 mg. Here we report the results of a randomized, double-blind, placebo-controlled trial that examined the antiviral activity, tolerability, and pharmacokinetics of single 5-mg/kg and 10-mg/kg intravenous infusions of PRO 140 in 31 treated subjects.

Anti-HIV-1 activity of weekly or biweekly treatment with subcutaneous PRO 140, a CCR5 monoclonal antibody.

Background: PRO 140 is a humanized CCR5 monoclonal antibody that has demonstrated potent antiviral activity when it is administered intravenously to adults infected with CCR5-tropic (R5) human immunodeficiency virus type 1 (HIV-1). This study is the first to evaluate subcutaneous administration.

Methods: A randomized, double-blind, placebo-controlled study was conducted among 44 subjects with HIV-1 RNA levels of >5000 copies/mL, CD4(+) cell counts of >300 cells/microL, no receipt of antiretroviral therapy for >or=12 weeks, and only R5 HIV-1 detectable.

Escape of HIV-1 from a small molecule CCR5 inhibitor is not associated with a fitness loss.

Fitness is a parameter used to quantify how well an organism adapts to its environment; in the present study, fitness is a measure of how well strains of human immunodeficiency virus type 1 (HIV-1) replicate in tissue culture. When HIV-1 develops resistance in vitro or in vivo to antiretroviral drugs such as reverse transcriptase or protease inhibitors, its fitness is often impaired. Here, we have investigated whether the development of resistance in vitro to a small molecule CCR5 inhibitor, AD101, has an associated fitness cost.

Natural variation in the V3 crown of human immunodeficiency virus type 1 affects replicative fitness and entry inhibitor sensitivity.

Natural polymorphisms in the heterogeneous human immunodeficiency virus type 1 (HIV-1) envelope glycoprotein may have an impact on both sensitivity to entry inhibitors and viral replicative fitness. Of significant interest is variation in the V3 crown due to its involvement in direct engagement with the coreceptor. Two positions in the crown (318 and 319) appear to be important in determining intrinsic susceptibility to multiple entry inhibitors.

HIV-1 clones resistant to a small molecule CCR5 inhibitor use the inhibitor-bound form of CCR5 for entry.

Human immunodeficiency virus type 1 (HIV-1) infection can be inhibited by small molecules that target the CCR5 coreceptor. Here, we describe some properties of clonal viruses resistant to one such inhibitor, SCH-D, using both chimeric, infectious molecular clones and Env-pseudotypes. Studies using combinations of CCR5 ligands, including small molecule inhibitors, monoclonal antibodies (MAbs) and chemokine derivatives such as PSC-RANTES, show that the fully SCH-D-resistant viruses enter target cells by using the SCH-D-bound form of CCR5.

A yeast recombination-based cloning system to produce chimeric HIV-1 viruses and express HIV-1 genes.

Differential phenotypes or properties of HIV-1 gene products in primary virus isolates are difficult to assess due to interference by the high degree of sequence variation across the entire genome. Thus, chimeric viruses provide a powerful tool to study the function of single gene products or genetic elements in the context of a neutral viral genomic backbone. In this chapter, we describe how to produce HIV-1 chimeric viruses utilizing a yeast-based homologous recombination cloning technique to insert env sequences first into a yeast cloning vector and then into the common pNL4-3 virus backbone.

Generation and properties of a human immunodeficiency virus type 1 isolate resistant to the small molecule CCR5 inhibitor, SCH-417690 (SCH-D).

We describe the generation of two genetically related human immunodeficiency virus type 1 (HIV-1) isolates highly (>20,000-fold) resistant to the small molecule CCR5 inhibitor, SCH-417690 (formerly SCH-D). Both viruses were cross-resistant to other small molecules targeting entry via CCR5, but they were inhibited by some MAbs against the same coreceptor on primary CD4+ T-cells. The resistant isolates remained sensitive to inhibitors of other stages of virus entry, and to replication inhibitors acting post-entry.

Differences in the fitness of two diverse wild-type human immunodeficiency virus type 1 isolates are related to the efficiency of cell binding and entry.

The ability of one primary human immunodeficiency virus type 1 (HIV-1) isolate to outcompete another in primary CD4+ human lymphoid cells appears to be mediated by the efficiency of host cell entry. This study was designed to test the role of entry on fitness of wild-type HIV-1 isolates (e.g.

Monitoring processed, mature Human Immunodeficiency Virus type 1 particles immediately following treatment with a protease inhibitor-containing treatment regimen.

Protease inhibitors (PIs) block HIV-1 maturation into an infectious virus particle by inhibiting the protease processing of gag and gag-pol precursor proteins. We have used a simple anti-HIV-1 p24 Western blot to monitor the processing of p55gag precursor into the mature p24 capsid immediately following the first dosage of a PI-containing treatment regimen. Evidence of PI activity was observed in plasma virus as early as 72 hours post treatment-initiation and was predictive of plasma viral RNA decrease at 4 weeks.

Relationships between infectious titer, capsid protein levels, and reverse transcriptase activities of diverse human immunodeficiency virus type 1 isolates.

Most studies on human immunodeficiency virus type 1 (HIV-1) replication kinetics or fitness must rely on a particular assay to initially standardize inocula from virus stocks. The most accurate measure of infectious HIV-1 titers involves a limiting dilution-infection assay and a calculation of the dose required for 50% infectivity of susceptible cells in tissue culture (TCID(50)). Surrogate assays are now commonly used to measure the amount of p24 capsid, the endogenous reverse transcriptase (RT) activity, or the amount of viral genomic RNA in virus particles.

Effect of a CCR5 inhibitor on viral loads in macaques dual-infected with R5 and X4 primate immunodeficiency viruses.

Human immunodeficiency virus type 1 (HIV-1) fusion with its target cells is initiated by sequential interactions between its envelope glycoprotein, CD4, and a co-receptor, usually CCR5 or CXCR4. Small molecules that bind to CCR5 and prevent its use by R5 HIV-1 strains are now being developed clinically as antiviral drugs. To test whether a block to CCR5 promotes the replication of viruses that enter cells via CXCR4 and are associated with accelerated disease progression, we administered a small molecule CCR5 inhibitor, CMPD 167, to three macaques dual-infected with both R5 (SIVmac251) and X4 (SHIV-89.