Chikungunya virus (CHIKV) is a reemerging alphavirus that causes a debilitating arthritic disease and infects millions of people and for which no specific treatment is available. Like many ...alphaviruses, the structural targets on CHIKV that elicit a protective humoral immune response in humans are poorly defined. Here we used phage display against virus-like particles (VLPs) to isolate seven human monoclonal antibodies (MAbs) against the CHIKV envelope glycoproteins E2 and E1. One MAb, IM-CKV063, was highly neutralizing (50% inhibitory concentration, 7.4 ng/ml), demonstrated high-affinity binding (320 pM), and was capable of therapeutic and prophylactic protection in multiple animal models up to 24 h postexposure. Epitope mapping using a comprehensive shotgun mutagenesis library of 910 mutants with E2/E1 alanine mutations demonstrated that IM-CKV063 binds to an intersubunit conformational epitope on domain A, a functionally important region of E2. MAbs against the highly conserved fusion loop have not previously been reported but were also isolated in our studies. Fusion loop MAbs were broadly cross-reactive against diverse alphaviruses but were nonneutralizing. Fusion loop MAb reactivity was affected by temperature and reactivity conditions, suggesting that the fusion loop is hidden in infectious virions. Visualization of the binding sites of 15 different MAbs on the structure of E2/E1 revealed that all epitopes are located at the membrane-distal region of the E2/E1 spike. Interestingly, epitopes on the exposed topmost and outer surfaces of the E2/E1 trimer structure were neutralizing, whereas epitopes facing the interior of the trimer were not, providing a rationale for vaccine design and therapeutic MAb development using the intact CHIKV E2/E1 trimer.
CHIKV is the most important alphavirus affecting humans, resulting in a chronic arthritic condition that can persist for months or years. In recent years, millions of people have been infected globally, and the spread of CHIKV to the Americas is now beginning, with over 100,000 cases occurring in the Caribbean within 6 months of its arrival. Our study reports on seven human MAbs against the CHIKV envelope, including a highly protective MAb and rarely isolated fusion loop MAbs. Epitope mapping of these MAbs demonstrates how some E2/E1 epitopes are exposed or hidden from the human immune system and suggests a structural mechanism by which these MAbs protect (or fail to protect) against CHIKV infection. Our results suggest that the membrane-distal end of CHIKV E2/E1 is the primary target for the humoral immune response to CHIKV, and antibodies targeting the exposed topmost and outer surfaces of the E2/E1 trimer determine the neutralizing efficacy of this response.
To exploit highly conserved and difficult drug targets, including multipass membrane proteins, monoclonal antibody discovery efforts increasingly rely on the advantages offered by divergent species ...such as rabbits, camelids, and chickens. Here, we provide an overview of antibody discovery technologies, analyze gaps in therapeutic antibodies that stem from the historic use of mice, and examine opportunities to exploit previously inaccessible targets through discovery now possible in alternate species. We summarize the clinical development of antibodies raised from divergent species, discussing how these animals enable robust immune responses against highly conserved binding sites and yield antibodies capable of penetrating functional pockets via long HCDR3 regions. We also discuss the value of pan-reactive molecules often produced by these hosts, and how these antibodies can be tested in accessible animal models, offering a faster path to clinical development.
Human arteries engineered in vitro McKee, J Andrew; Banik, Soma SR; Boyer, Matthew J ...
EMBO reports,
June 2003, Letnik:
4, Številka:
6
Journal Article
Recenzirano
Odprti dostop
There is a pressing need to develop methods to engineer small‐calibre arteries for bypass surgery. We hypothesized that the rate‐limiting step that has thwarted previous attempts to engineer such ...vessels from non‐neonatal tissues is the limited proliferative capacity of smooth muscle cells (SMCs), which are the main cellular component of these vessels. Ectopic expression of the human telomerase reverse transcriptase subunit (hTERT) has been shown recently to extend the lifespan of certain human cells. We therefore introduced hTERT into human SMCs and found that the resulting cells proliferated far beyond their normal lifespan but retained characteristics of normal control SMCs. Importantly, using these non‐neonatal SMCs, we were able to engineer mechanically robust human vessels, a crucial step towards creating arteries of clinical value for bypass surgery.
Blood vessels engineered from human cells Poh, Melissa; Boyer, Matthew; Solan, Amy ...
The Lancet (British edition),
06/2005, Letnik:
365, Številka:
9477
Journal Article
Recenzirano
Tissue engineering has made considerable progress in the past decade, but advances have stopped short of clinical application for most tissues. We postulated that an obstacle in engineering human ...tissues is the limited replicative capacity of adult somatic cells. To test this hypothesis, the effectiveness of telomerase expression to extend cellular lifespan was assessed in a model of human vascular tissue engineering. Telomerase expression in vascular cells isolated from elderly patients enabled the successful culture of engineered autologous blood vessels. Engineered vessels may one day provide a source of bypass conduit for patients with atherosclerotic disease.
The addition of telomeric repeats to chromosome ends by the enzyme telomerase is a highly orchestrated process. Although much
is known regarding telomerase catalytic activity in vitro , less is known ...about how this activity is regulated in vivo to ensure proper telomere elongation. One protein that appears to be involved in negatively regulating telomerase function
in vivo is PinX1 because overexpression of PinX1 inhibits telomerase activity and causes telomere shortening. To understand the nature
of this repression, we characterized the interactions among PinX1 and the core components of telomerase, the human telomerase
reverse transcriptase (hTERT) and associated human telomerase RNA (hTR). We now show that in vitro PinX1 binds directly to the hTERT protein subunit, primarily to the hTR-binding domain, as well as to the hTR subunit. However,
in a cellular context, the association of PinX1 with hTR is dependent on the presence of hTERT. Taken together, we suggest
that PinX1 represses telomerase activity in vivo by binding to the assembled hTERT·hTR complex.
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