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Plaque reduction assays for human and simian immunodeficiency virus neutralisation.

Nordqvist, Anna and Fenyö, Eva Maria LU (2005) In Human Retrovirus Protocols 1. p.273-285
Abstract
Research on HIV vaccines, as well as studies on HIV pathogenesis in human and SIV in the macaque model, require the availability of simple and standardized assays for quantification of neutralizing antibodies to primary virus isolates. We have recently developed and standardized assays using human cell lines engineered to express CD4 and co-receptors for HIV and SIV entry. One cell line originated from a glioma (U87) and the other from an osteosarcoma (HOS). Both cell lines and their derivatives form monolayer cultures, a prerequisite for counting plaques. HIV-infected U87.CD4-CCR5 or -CXCR4 cells form syncytia, that is, plaques that can be stained with hematoxylin and enumerated by light microscopy. In addition to CD4 and co-receptors... (More)
Research on HIV vaccines, as well as studies on HIV pathogenesis in human and SIV in the macaque model, require the availability of simple and standardized assays for quantification of neutralizing antibodies to primary virus isolates. We have recently developed and standardized assays using human cell lines engineered to express CD4 and co-receptors for HIV and SIV entry. One cell line originated from a glioma (U87) and the other from an osteosarcoma (HOS). Both cell lines and their derivatives form monolayer cultures, a prerequisite for counting plaques. HIV-infected U87.CD4-CCR5 or -CXCR4 cells form syncytia, that is, plaques that can be stained with hematoxylin and enumerated by light microscopy. In addition to CD4 and co-receptors (most often used CCR5 and CXCR6 by SIV), GHOST(3) cells have been engineered to express the green fluorescent protein following virus infection. Infected cells show green fluorescence and can be enumerated by fluorescence microscopy. Neutralization is determined by the ability of a serum to reduce the number of plaque-forming units (PFU) relative to controls exposed to medium or negative serum. Both assays are run in microtiter format and neutralization is evaluated after 3 d. Intra-assay variation has been used for estimation of the cutoff for neutralization. Testing 15 serum-virus combinations in the U87.CD4 assay and four serum-virus combinations in the GHOST(3) assay revealed that standard deviation of differences ranged from 9.1% to 9.9% in the two assays. This allowed the use of a cutoff >3 SD; that is, 30% neutralization. Virus titration experiments showed that neutralization results were dependent on virus dose and therefore the neutralization assays should be performed with a virus dose of 10–100 PFU/well. The assays have high specificity and reproducibility, and are simple and sensitive high-throughput assays. (Less)
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author
organization
publishing date
type
Chapter in Book/Report/Conference proceeding
publication status
published
subject
keywords
engineered cell lines, co-receptor use, neutralization assays, plaque assays, HIV, SIV
in
Human Retrovirus Protocols
editor
Zhu, Tuofu and
volume
1
pages
273 - 285
publisher
Humana Press
external identifiers
  • scopus:24944554617
ISSN
1940-6029
1064-3745
ISBN
978-1-58829-495-1
DOI
10.1385/1-59259-907-9:273
language
English
LU publication?
yes
id
f8d27c53-beec-4725-8f26-1e902011293f (old id 1134150)
date added to LUP
2008-06-18 13:07:41
date last changed
2017-01-01 04:40:10
@inbook{f8d27c53-beec-4725-8f26-1e902011293f,
  abstract     = {Research on HIV vaccines, as well as studies on HIV pathogenesis in human and SIV in the macaque model, require the availability of simple and standardized assays for quantification of neutralizing antibodies to primary virus isolates. We have recently developed and standardized assays using human cell lines engineered to express CD4 and co-receptors for HIV and SIV entry. One cell line originated from a glioma (U87) and the other from an osteosarcoma (HOS). Both cell lines and their derivatives form monolayer cultures, a prerequisite for counting plaques. HIV-infected U87.CD4-CCR5 or -CXCR4 cells form syncytia, that is, plaques that can be stained with hematoxylin and enumerated by light microscopy. In addition to CD4 and co-receptors (most often used CCR5 and CXCR6 by SIV), GHOST(3) cells have been engineered to express the green fluorescent protein following virus infection. Infected cells show green fluorescence and can be enumerated by fluorescence microscopy. Neutralization is determined by the ability of a serum to reduce the number of plaque-forming units (PFU) relative to controls exposed to medium or negative serum. Both assays are run in microtiter format and neutralization is evaluated after 3 d. Intra-assay variation has been used for estimation of the cutoff for neutralization. Testing 15 serum-virus combinations in the U87.CD4 assay and four serum-virus combinations in the GHOST(3) assay revealed that standard deviation of differences ranged from 9.1% to 9.9% in the two assays. This allowed the use of a cutoff >3 SD; that is, 30% neutralization. Virus titration experiments showed that neutralization results were dependent on virus dose and therefore the neutralization assays should be performed with a virus dose of 10–100 PFU/well. The assays have high specificity and reproducibility, and are simple and sensitive high-throughput assays.},
  author       = {Nordqvist, Anna and Fenyö, Eva Maria},
  editor       = {Zhu, Tuofu},
  isbn         = {978-1-58829-495-1},
  issn         = {1940-6029},
  keyword      = {engineered cell lines,co-receptor use,neutralization assays,plaque assays,HIV,SIV},
  language     = {eng},
  pages        = {273--285},
  publisher    = {Humana Press},
  series       = {Human Retrovirus Protocols},
  title        = {Plaque reduction assays for human and simian immunodeficiency virus neutralisation.},
  url          = {http://dx.doi.org/10.1385/1-59259-907-9:273},
  volume       = {1},
  year         = {2005},
}