Expression, purification, and characterization of recombinant cyanovirin-N for vaginal anti-HIV microbicide development

Colleluori, Diana M.; Tien, Deborah; Kang, Feirong; Pagliei, Tara; Kuss, Ryan; McCormick, Timothy; Watson, Karen; McFadden, Karyn; Chaiken, Irwin M.; Buckheit, Robert W.; Romano, Joseph

doi:10.1016/j.pep.2004.10.009

Cited by 51 publications

(42 citation statements)

References 16 publications

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“…Initial characterizations of the CV-N proteins were conducted by assaying activity against the clade B HIV strain SC422661.8. WT CV-N showed half-maximal neutralization at concentrations (IC 50 s) between 1.0 and 9.4 nM (0.012 to 0.12 μg∕mL) over 30 independent trials, with an average of 4.4 AE 2.6 nM (0.054 AE 0.032 μg∕mL), consistent with published values (20,(23)(24)(25).…”

Section: Resultssupporting

confidence: 86%

“…WT CV-N itself has a high affinity for gp120 (1,23), but by doubling the number of carbohydrate binding sites in the CVN 2 variants, the increase in avidity may prevent possible dissociation and escape of the virus. As shown by our knockout studies, CVN 2 L0 variants with more functional binding sites were significantly more potent at neutralizing HIV, indicating that higher avidity translates to greater potency.…”

Section: Discussionmentioning

confidence: 99%

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Designed oligomers of cyanovirin-N show enhanced HIV neutralization

Keeffe¹,

Gnanapragasam²,

Gillespie³

et al. 2011

Proc. Natl. Acad. Sci. U.S.A.

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Cyanovirin-N (CV-N) is a small, cyanobacterial lectin that neutralizes many enveloped viruses, including human immunodeficiency virus type I (HIV-1). This antiviral activity is attributed to two homologous carbohydrate binding sites that specifically bind high mannose glycosylation present on envelope glycoproteins such as HIV-1 gp120. We created obligate CV-N oligomers to determine whether increasing the number of binding sites has an effect on viral neutralization. A tandem repeat of two CV-N molecules (CVN 2 ) increased HIV-1 neutralization activity by up to 18-fold compared to wild-type CV-N. In addition, the CVN 2 variants showed extensive cross-clade reactivity and were often more potent than broadly neutralizing anti-HIV antibodies. The improvement in activity and broad cross-strain HIV neutralization exhibited by these molecules holds promise for the future therapeutic utility of these and other engineered CV-N variants.

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Section: Resultssupporting

confidence: 86%

Section: Discussionmentioning

confidence: 99%

Designed oligomers of cyanovirin-N show enhanced HIV neutralization

Keeffe¹,

Gnanapragasam²,

Gillespie³

et al. 2011

Proc. Natl. Acad. Sci. U.S.A.

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“…The final concentration was determined using absorbance at 280 nm and an extinction coefficient at 280 nm of 39,740 M Ϫ1 cm Ϫ1 . Cyanovirin-N and gp120 were produced in E. coli and CHO (Chinese hamster ovary) cells, respectively, as previously reported (19)(20)(21).…”

Section: Allmentioning

confidence: 99%

Chimeric Cyanovirin-MPER Recombinantly Engineered Proteins Cause Cell-Free Virolysis of HIV-1

Contarino

Bastian

Sundaram

et al. 2013

Antimicrob Agents Chemother

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Human immunodeficiency virus (HIV) is the primary etiologic agent responsible for the AIDS pandemic. In this work, we used a chimeric recombinant protein strategy to test the possibility of irreversibly destroying the HIV-1 virion using an agent that simultaneously binds the Env protein and viral membrane. We constructed a fusion of the lectin cyanovirin-N (CVN) and the gp41 membrane-proximal external region (MPER) peptide with a variable-length (Gly 4 Ser) x linker (where x is 4 or 8) between the C terminus of the former and N terminus of the latter. The His-tagged recombinant proteins, expressed in BL21(DE3)pLysS cells and purified by immobilized metal affinity chromatography followed by gel filtration, were found to display a nanomolar efficacy in blocking BaL-pseudotyped HIV-1 infection of HOS.T4.R5 cells. This antiviral activity was HIV-1 specific, since it did not inhibit cell infection by vesicular stomatitis virus (VSV) or amphotropic-murine leukemia virus. Importantly, the chimeric proteins were found to release intraviral p24 protein from both BaL-pseudotyped HIV-1 and fully infectious BaL HIV-1 in a dose-dependent manner in the absence of host cells. The addition of either MPER or CVN was found to outcompete this virolytic effect, indicating that both components of the chimera are required for virolysis. The finding that engaging the Env protein spike and membrane using a chimeric ligand can destabilize the virus and lead to inactivation opens up a means to investigate virus particle metastability and to evaluate this approach for inactivation at the earliest stages of exposure to virus and before host cell encounter. HIV-1 is a global health epidemic that leads to the deaths of over two million people annually through eventual progression into AIDS. Highly active antiretroviral therapy (HAART) (1) has proven effective at delaying the onset of AIDS in HIV-positive individuals but does not provide a cure for the infection itself. Despite decades of research, no vaccine-or microbicide-based approach for preventing transmission of HIV-1 to noninfected individuals is available. While the leading microbicidal candidate is based on tenofovir, a reverse transcriptase inhibitor (2), there are no reported examples of FDA-approved agents or combinations of agents that directly and specifically destroy mature HIV-1 particles before they gain entry into a target cell (3). Therefore, there is an opportunity to broaden the number of microbicidal candidates by rational design of agents that specifically prevent HIV-1 entry and irreversibly destroy infectious virus.A mature HIV-1 virion is an approximately 1-aL-sized (4) bilayer-enveloped packet of cytoplasm stolen from the cell from which the virion budded, surrounding the RNA-containing nucleocapsid. HIV-1 enters target cells via interactions between the viral envelope protein spike, Env, with the surface-expressed CD4 receptor and a chemokine coreceptor (CCR5 or CXCR4) (5, 6). The Env spike is a metastable heterotrimeric protein complex of three transmembrane gp41...

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“…For global distribution, a topical microbicide should also be inexpensive to produce and stable without refrigeration. CV-N, as a conventional protein-based microbicide candidate, is expected to be costly to produce (12) and relatively unstable at ambient temperatures, making it an unrealistic product, particularly in economically disadvantaged regions. In addition, protein-based microbicides, as recently exemplified by a gel formulation of CV-N used in a macaque HIV vaginal transmission model (43), may also require administration of relatively high concentrations due to the relatively short half-life and/or rapid clearance of these molecules (43).…”

Section: Discussionmentioning

confidence: 99%