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PLoS One. 2014 Oct 3;9(10):e109196. doi: 10.1371/journal.pone.0109196. eCollection 2014.

A mammalian cell based FACS-panning platform for the selection of HIV-1 envelopes for vaccine development.

Author information

1
Institute of Medical Microbiology and Hygiene, University Regensburg, Regensburg, Bavaria, Germany.

Abstract

An increasing number of broadly neutralizing monoclonal antibodies (bnMAb) against the HIV-1 envelope (Env) protein has been discovered recently. Despite this progress, vaccination efforts with the aim to re-elicit bnMAbs that provide protective immunity have failed so far. Herein, we describe the development of a mammalian cell based FACS-panning method in which bnMAbs are used as tools to select surface-exposed envelope variants according to their binding affinity. For that purpose, an HIV-1 derived lentiviral vector was developed to infect HEK293T cells at low multiplicity of infection (MOI) in order to link Env phenotype and genotype. For proof of principle, a gp145 Env model-library was established in which the complete V3 domain was substituted by five strain specific V3 loop sequences with known binding affinities to nMAb 447-52D, respectively. Env genes were recovered from selected cells by PCR, subcloned into a lentiviral vector (i) to determine and quantify the enrichment nMAb binders and (ii) to generate a new batch of transduction competent particles. After 2 selection cycles the Env variant with highest affinity was enriched 20-fold and represented 80% of the remaining Env population. Exploiting the recently described bnMAbs, this procedure might prove useful in selecting Env proteins from large Env libraries with the potential to elicit bnMAbs when used as vaccine candidates.

PMID:
25279768
PMCID:
PMC4184847
DOI:
10.1371/journal.pone.0109196
[Indexed for MEDLINE]
Free PMC Article

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