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Analysis of SIVmac Envelope-Specific Antibodies Selected Through Phage Display.

ABSTRACT: We have constructed a single chain fragment variable (scFv) phage display library from a simian immunodeficiency virus (SIV)-infected rhesus macaque that developed unusually high-titer neutralizing antibody responses against tier-3, neutralization-resistant SIVmac239. The library was screened using trimeric (gp140) and monomeric (gp120) forms of the SIVmac239 envelope (Env) glycoprotein. We also cloned variable-heavy and variable-light (VH-VL) antibody fragments from seven previously described rhesus macaque B-cell lines (BLCLs) that produce SIV gp120-specific monoclonal antibodies (mAbs). Thirty-two gp140-specific mAbs were selected along with 20 gp120-specific ones. gp120-specific mAbs were only from the VH4 family, while gp41-specific mAbs were primarily from VH1, followed by VH4 and VH3. Rhesus macaque BLCL-derived mAbs belonged primarily to the VH4 family of antibodies followed by VH3 and a smaller number of VH1s. A preferential VH combination with V? light chain was observed with phage display-selected SIV Env-specific mAbs (gp120 and gp140), but not with BLCL-derived antibodies or the unpanned library. None of the tested antibodies had detectable neutralizing activity against tier-3 SIVmac239. The majority of gp120-specifc mAbs potently neutralized tier-1 SIVmac316 with 50% inhibitory concentration (IC50) values below 1??g/ml. For gp140-specific antibodies, which were all specific for the gp41-subunit, 2 out of 11 tested neutralized SIVmac316 (IC50 of 7 and 5??g/ml, respectively). These data suggest an order of preferential VH segment usage for SIV-specific antibodies in rhesus macaques. These antibodies will be useful in assessing the contribution of non-neutralizing antibodies to inhibition of SIV infection in vitro and in vivo.

SUBMITTER: Ita S

PROVIDER: S-EPMC5749048 | biostudies-literature | 2017 Aug

REPOSITORIES: biostudies-literature

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Analysis of SIVmac Envelope-Specific Antibodies Selected Through Phage Display.

Ita Sergio S Agostinho Mayara R MR Sullivan Katherine K Yub Han Seung S Akleh Rana R Johnson Welkin E WE Fofana Ismael Ben F IBF

AIDS research and human retroviruses 20170216 8

We have constructed a single chain fragment variable (scFv) phage display library from a simian immunodeficiency virus (SIV)-infected rhesus macaque that developed unusually high-titer neutralizing antibody responses against tier-3, neutralization-resistant SIVmac239. The library was screened using trimeric (gp140) and monomeric (gp120) forms of the SIVmac239 envelope (Env) glycoprotein. We also cloned variable-heavy and variable-light (VH-VL) antibody fragments from seven previously described r ...[more]

PMID: 28075174

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Project description:BackgroundSingle cell genomics has revolutionized microbial sequencing, but complete coverage of genomes in complex microbiomes is imperfect due to enormous variation in organismal abundance and amplification bias. Empirical methods that complement rapidly improving bioinformatic tools will improve characterization of microbiomes and facilitate better genome coverage for low abundance microbes.MethodsWe describe a new approach to sequencing individual species from microbiomes that combines antibody phage display against intact bacteria with fluorescence activated cell sorting (FACS). Single chain (scFv) antibodies are selected using phage display against a bacteria or microbial community, resulting in species-specific antibodies that can be used in FACS for relative quantification of an organism in a community, as well as enrichment or depletion prior to genome sequencing.ResultsWe selected antibodies against Lactobacillus acidophilus and demonstrate a FACS-based approach for identification and enrichment of the organism from both laboratory-cultured and commercially derived bacterial mixtures. The ability to selectively enrich for L. acidophilus when it is present at a very low abundance (<0.2%) leads to complete (>99.8%) de novo genome coverage whereas the standard single-cell sequencing approach is incomplete (<68%). We show that specific antibodies can be selected against L. acidophilus when the monoculture is used as antigen as well as when a community of 10 closely related species is used demonstrating that in principal antibodies can be generated against individual organisms within microbial communities.ConclusionsThe approach presented here demonstrates that phage-selected antibodies against bacteria enable identification, enrichment of rare species, and depletion of abundant organisms making it tractable to virtually any microbe or microbial community. Combining antibody specificity with FACS provides a new approach for characterizing and manipulating microbial communities prior to genome sequencing.

Dataset Information

Analysis of SIVmac Envelope-Specific Antibodies Selected Through Phage Display.

Publications

Analysis of SIVmac Envelope-Specific Antibodies Selected Through Phage Display.

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