Rapid Development of a Registry to Accelerate COVID-19 Vaccine Clinical Trials.

Background: The unprecedented scientific response to the SARS-Cov-2 pandemic in 2020 required the rapid development and activation of extensive clinical trial networks to study vaccines and therapeutics. The COVID-19 Prevention Network (CoVPN) coordinated hundreds of sites conducting phase 2 and 3 clinical trials of vaccines and antibody therapeutics. To facilitate these clinical trials, the CoVPN Volunteer Screening Registry (VSR) was created to collect volunteer information at scale, identify volunteers at risk of COVID-19 who met enrollment criteria, distribute candidates across clinical trial sites, and enable monitoring of volunteering and enrollment progress.

Post-COVID symptom profiles and duration in a global convalescent COVID-19 observational cohort: Correlations with demographics, medical history, acute COVID-19 severity and global region.

Background: Post-COVID conditions are characterised by persistent symptoms that negatively impact quality of life after SARS-CoV-2 diagnosis. While post-COVID risk factors and symptoms have been extensively described in localised regions, especially in the global north, post-COVID conditions remain poorly understood globally. The global, observational cohort study HVTN 405/HPTN 1901 characterises the convalescent course of SARS-CoV-2 infection among adults in North and South America and Africa.

Cellular and humoral responses to an HIV DNA prime by electroporation boosted with recombinant vesicular stomatitis virus expressing HIV subtype C Env in a randomized controlled clinical trial.

Background: HIV subtypes B and C together account for around 60% of HIV-1 cases worldwide. We evaluated the safety and immunogenicity of a subtype B DNA vaccine prime followed by a subtype C viral vector boost.

Methods: Fourteen healthy adults received DNA plasmid encoding HIV-1 subtype B nef/tat/vif and env (n = 11) or placebo (n = 3) intramuscularly (IM) via electroporation (EP) at 0, 1, and 3 months, followed by IM injection of recombinant vesicular stomatitis virus encoding subtype C Env or placebo at 6 and 9 months.

Neutralizing antibody responses over time in demographically and clinically diverse individuals recovered from SARS-CoV-2 infection in the United States and Peru: A cohort study.

Background: People infected with Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) experience a wide range of clinical manifestations, from asymptomatic and mild illness to severe illness and death, influenced by age and a variety of comorbidities. Neutralizing antibodies (nAbs) are thought to be a primary immune defense against the virus. Large, diverse, well-characterized cohorts of convalescent individuals provide standardized values to benchmark nAb responses to past SARS-CoV-2 infection and define potentially protective levels of immunity.

Applications of Lipid Nanodiscs for the Study of Membrane Proteins by Surface Plasmon Resonance.

Methods for the initial steps of surface plasmon resonance analysis of membrane proteins incorporated in lipid nanodiscs are described. Several types of Biacore sensor chips are available and require distinct strategies to immobilize proteonanodiscs on the chip surface. The procedures for immobilization on three of these chips (NTA, antibody coupled CM5, and L1) are described in this unit and results are demonstrated for a model system with cytochrome P4503A4 (CYP3A4) in nanodiscs binding to a polyclonal anti-CYP3A4 antibody.

Poliovirus infection transiently increases COPII vesicle budding.

Poliovirus (PV) requires membranes of the host cell's secretory pathway to generate replication complexes (RCs) for viral RNA synthesis. Recent work identified the intermediate compartment and the Golgi apparatus as the precursors of the replication "organelles" of PV (N. Y.

Transport vesicle uncoating: it's later than you think.

Transport vesicle coat proteins play active roles in vesicle cargo sorting as well as membrane deformation and fission during vesicle biogenesis. For years, it was assumed that this was the extent of the coats' function and that the coats depolymerized immediately after vesicle budding, leaving the exposed fusion machinery free to find, dock, and fuse with the proper target membrane. Recently, however, it has become increasingly clear that the coat remains on transport vesicles during their post-budding life and in fact helps properly pair up the vesicle with its intended target membrane.

An antibody directed against the fusion peptide of Junin virus envelope glycoprotein GPC inhibits pH-induced membrane fusion.

The arenavirus envelope glycoprotein (GPC) initiates infection in the host cell through pH-induced fusion of the viral and endosomal membranes. As in other class I viral fusion proteins, this process proceeds through a structural reorganization in GPC in which the ectodomain of the transmembrane fusion subunit (G2) engages the host cell membrane and subsequently refolds to form a highly stable six-helix bundle structure that brings the two membranes into apposition for fusion. Here, we describe a G2-directed monoclonal antibody, F100G5, that prevents membrane fusion by binding to an intermediate form of the protein on the fusion pathway.

Bitopic membrane topology of the stable signal peptide in the tripartite Junín virus GP-C envelope glycoprotein complex.

The stable signal peptide (SSP) of the GP-C envelope glycoprotein of the Junín arenavirus plays a critical role in trafficking of the GP-C complex to the cell surface and in its membrane fusion activity. SSP therefore may function on both sides of the lipid membrane. In this study, we have investigated the membrane topology of SSP by confocal microscopy of cells treated with the detergent digitonin to selectively permeabilize the plasma membrane.

Inhibitors of protein-disulfide isomerase prevent cleavage of disulfide bonds in receptor-bound glycoprotein 120 and prevent HIV-1 entry.

We previously reported that monoclonal antibodies to protein-disulfide isomerase (PDI) and other membrane-impermeant PDI inhibitors prevented HIV-1 infection. PDI is present at the surface of HIV-1 target cells and reduces disulfide bonds in a model peptide attached to the cell membrane. Here we show that soluble PDI cleaves disulfide bonds in recombinant envelope glycoprotein gp120 and that gp120 bound to the surface receptor CD4 undergoes a disulfide reduction that is prevented by PDI inhibitors.

Antibody binding and neutralization of primary and T-cell line-adapted isolates of human immunodeficiency virus type 1.

The relative resistance of human immunodeficiency virus type 1 (HIV-1) primary isolates (PIs) to neutralization by a wide range of antibodies remains a theoretical and practical barrier to the development of an effective HIV vaccine. One model to account for the differential neutralization sensitivity between Pls and laboratory (or T-cell line-adapted [TCLA]) strains of HIV suggests that the envelope protein (Env) complex is made more accessible to antibody binding as a consequence of adaptation to growth in established cell lines. Here, we revisit this question using genetically related PI and TCLA viruses and molecularly cloned env genes.

Turning a corner on HIV neutralization?

HIV vaccine development has been hampered by the inability of conventional immunogens to elicit antibodies capable of neutralizing primary isolates of the virus. Recent studies using 'fusion-competent' immunogens that capture transitional intermediate structures of the functioning envelope protein suggest that this goal may now be achievable.

Cyclophilin C-associated protein: a normal secreted glycoprotein that down-modulates endotoxin and proinflammatory responses in vivo.

Mouse cyclophilin C-associated protein (CyCAP) is a member of the scavenger-receptor cysteine-rich domain superfamily and is 69% identical to the human Mac-2 binding protein. Here, we show that CyCAP is a widely expressed secreted glycoprotein that modulates the host response to endotoxin. Gene-targeted CyCAP-deficient mice are more sensitive to the lethal effects of endotoxin.

Fusion-competent vaccines: broad neutralization of primary isolates of HIV.

Current recombinant human immunodeficiency virus (HIV) gp120 protein vaccine candidates are unable to elicit antibodies capable of neutralizing infectivity of primary isolates from patients. Here, "fusion-competent" HIV vaccine immunogens were generated that capture the transient envelope-CD4-coreceptor structures that arise during HIV binding and fusion. In a transgenic mouse immunization model, these formaldehyde-fixed whole-cell vaccines elicited antibodies capable of neutralizing infectivity of 23 of 24 primary HIV isolates from diverse geographic locations and genetic clades A to E.

Continued utilization of CCR5 coreceptor by a newly derived T-cell line-adapted isolate of human immunodeficiency virus type 1.

The differential use of CC chemokine receptor 5 (CCR5) and CXC chemokine receptor 4 (CXCR4) may be intimately involved in the transmission and progression of human immunodeficiency virus infection. Changes in coreceptor utilization have also been noted upon adaptation of primary isolates (PI) to growth in established T-cell lines. All of the T-cell line-adapted (TCLA) viruses studied to date utilize CXCR4 but not CCR5.

Coreceptor utilization by human immunodeficiency virus type 1 is not a primary determinant of neutralization sensitivity.

We have examined the relationship between coreceptor utilization and sensitivity to neutralization in a primary isolate of human immunodeficiency virus type 1 and its T-cell line-adapted (TCLA) derivative. We determined that adaptation of the primary-isolate (PI) virus 168P results in the loss of the unique capacity of PI viruses to utilize the CCR5 coreceptor and in the acquisition by the TCLA 168C virus of sensitivity to neutralization by V3-directed monoclonal antibodies (MAbs). In experiments wherein infection by 168P is directed via either the CCR5 or the CXCR4 pathway, we demonstrate that the virus, as well as pseudotyped virions bearing a molecularly cloned 168P envelope protein, remains refractory to neutralization by MAbs 257-D, 268-D, and 50.

Cloning and characterization of cyclophilin C-associated protein: a candidate natural cellular ligand for cyclophilin C.

We report the protein purification and the cloning and characterization of a cDNA encoding the proteins that bind with high affinity to cyclophilin C (Cyp-C) in the absence of cyclosporin A. Transfection of this cDNA into COS cells directs the production of a glycoprotein of 77 kDa that binds to Cyp-C in the absence, but not the presence, of cyclosporin A. Homology comparisons reveal that this protein and gene, termed CyCAP for Cyp-C-associated protein, possess a cysteine-rich domain (scavenger receptor cysteine-rich domain) found in a variety of cell-surface molecules; the rest of the sequence is apparently specific.

Molecular cloning of two types of GAP complementary DNA from human placenta.

The ras p21 GTPase-activating protein (GAP) was purified from human placental tissue. Internal amino acid sequence was obtained from this 120,000-dalton protein and, by means of this sequence, two types of complementary DNA clones were isolated and characterized. One type encoded GAP with a predicted molecular mass of 116,000 daltons and 96% identity with bovine GAP.

A cytoplasmic protein stimulates normal N-ras p21 GTPase, but does not affect oncogenic mutants.

The role of guanine nucleotides in ras p21 function was determined by using the ability of p21 protein to induce maturation of Xenopus oocytes as a quantitative assay for biological activity. Two oncogenic mutant human N-ras p21 proteins, Asp12 and Val12, actively induced maturation, whereas normal Gly12 p21 was relatively inactive in this assay. Both mutant proteins were found to be associated with guanosine triphosphate (GTP) in vivo.

Biochemical and biological properties of the human N-ras p21 protein.

We characterized the normal (Gly-12) and two mutant (Asp-12 and Val-12) forms of human N-ras proteins produced by Escherichia coli. No significant differences were found between normal and mutant p21 proteins in their affinities for GTP or GDP. Examination of GTPase activities revealed significant differences between the mutant p21s: the Val-12 mutant retained 12% of wild-type GTPase activity, whereas the Asp-12 mutant retained 43%.

Inhibition of protein synthesis stimulates the transcription of human beta-interferon genes in Chinese hamster ovary cells.

Using Chinese hamster ovary (CHO) cells transfected with a plasmid carrying the human beta-interferon gene, we find that inhibitors of protein synthesis, in the absence of any other inducer, stimulate the production of interferon RNA; this effect is maintained in cells in which the plasmid sequences have been amplified 25- to 50-fold. Nuclear transcription assays show that a major effect of cycloheximide is to increase the rate of transcription of the interferon gene. This contradicts the generally accepted explanation that inhibitors of protein synthesis augment interferon production by stabilizing interferon mRNA.

Inducible expression of amplified human beta interferon genes in CHO cells.

Plasmid DNA containing the human beta-interferon (IFN-beta) gene and mouse dihydrofolate reductase cDNA was transfected into dihydrofolate reductase-negative Chinese hamster ovary cells. Dihydrofolate reductase-positive transformants were obtained, and cells containing amplified copies of mouse dihydrofolate reductase were selected by exposure to increasing methotrexate concentrations. These cells were found to express high levels of human IFN-beta after polyriboinosinic acid-polyribocytidylic acid superinduction or NDV infection; this was a result of coamplification of the IFN-beta gene.