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Effects of galectins and antibodies in HIV infection; Novel Assays

Sheik-Khalil, Enas LU (2015) In Lund University, Faculty of Medicine Doctoral Dissertation Series 2015:36.
Abstract
The high variability of the HIV envelope glycoproteins (Env), and their heavy glycan coating, contributes to the limited host immune

control. Still, broadly neutralizing antibodies (bnAbs) are found in some chronically infected HIV-infected individuals, which has

spurred the research on antibody-based vaccines. An important tool in detecting and studying bnAbs, are neutralization assays. Here

we developed an image-based, high-content automated version of a plaque reduction (PR) assay, which uses green fluorescent protein

expression as a reporter of HIV infection. This permitted simultaneous detection of antibodies mediating neutralization and

inhibition of virus induced cell-cell fusion. In a... (More)
The high variability of the HIV envelope glycoproteins (Env), and their heavy glycan coating, contributes to the limited host immune

control. Still, broadly neutralizing antibodies (bnAbs) are found in some chronically infected HIV-infected individuals, which has

spurred the research on antibody-based vaccines. An important tool in detecting and studying bnAbs, are neutralization assays. Here

we developed an image-based, high-content automated version of a plaque reduction (PR) assay, which uses green fluorescent protein

expression as a reporter of HIV infection. This permitted simultaneous detection of antibodies mediating neutralization and

inhibition of virus induced cell-cell fusion. In a multicenter study, Neutnet II, the assay compared well with other neutralization

assays and was suggested to be an alternative to the traditional peripheral blood monocyte (PBMC)-based assay and the TZMbl assay.

The glycans of Env can also take part in HIV host cell adhesion and infection, via host glycan-binding protein, such as galectins. To

explore this, we examined the interaction of gp120 (the surface Env protein) with a panel of galectins, by adapting the fluorescent

anisotropy (FA) assay to microscale. Galectin-8, a galectin with two carbohydrate recognition domains (CRDs), had high affinity for

gp120 as well as the HIV receptor CD4. The N-terminal CRD mediated the strongest interaction with gp120. The results of the FAassay

correlated well with binding of whole virions screened in another assay against the same panel of galectins, now immobilized on

beads. In the PR assay described above, here used as an infectivity assay, added intact galectin-8 enhanced infectivity of some HIV-1-

strains, while this was not seen with the N-CRD, demonstrating that both CRDs of galectin-8 are required for the effect on

infectivity. The enhancement effect mediated by galectin-8 was most pronounced with HIV-1 isolates obtained during the relative

immune competent chronic phase, as compared to viruses isolated after AIDS onset. Hence, galectin-8 binding carbohydrate motifs

on Env appear to be altered at severe immunodeficiency, adding to the knowledge on the evolution of Env glycosylation patterns

related to HIV pathogenesis. These results add to the basic knowledge of virus-host interactions, which hopefully could be used for

identification of antibodies and galectin-inhibitors effective in HIV prophylactic interventions. (Less)
Please use this url to cite or link to this publication:
author
supervisor
opponent
  • professor Olofsson, Sigvard, Göteborgs Universitet
organization
publishing date
type
Thesis
publication status
published
subject
keywords
HIV-1, galectin-1, galectin-8, neutralizing antibodies, cell-cell fusion, Microscale anisotropy assay, virus binding assay, glycosylation, gp120, Automated plaque reduction assay, CellProfiler, NeutNet
in
Lund University, Faculty of Medicine Doctoral Dissertation Series
volume
2015:36
pages
181 pages
publisher
Microbiology, Immunology and Glycobiology, MIG, Lund University
defense location
Belfragesalen, D15, BMC, Klinikgatan 32, Lund
defense date
2015-04-17 09:00
ISSN
1652-8220
ISBN
978-91-7619-115-6
language
English
LU publication?
yes
id
28230acc-2e49-4e80-92a6-4ff26eaf0a4a (old id 5212383)
date added to LUP
2015-03-27 11:52:21
date last changed
2016-09-19 08:44:50
@phdthesis{28230acc-2e49-4e80-92a6-4ff26eaf0a4a,
  abstract     = {The high variability of the HIV envelope glycoproteins (Env), and their heavy glycan coating, contributes to the limited host immune<br/><br>
control. Still, broadly neutralizing antibodies (bnAbs) are found in some chronically infected HIV-infected individuals, which has<br/><br>
spurred the research on antibody-based vaccines. An important tool in detecting and studying bnAbs, are neutralization assays. Here<br/><br>
we developed an image-based, high-content automated version of a plaque reduction (PR) assay, which uses green fluorescent protein<br/><br>
expression as a reporter of HIV infection. This permitted simultaneous detection of antibodies mediating neutralization and<br/><br>
inhibition of virus induced cell-cell fusion. In a multicenter study, Neutnet II, the assay compared well with other neutralization<br/><br>
assays and was suggested to be an alternative to the traditional peripheral blood monocyte (PBMC)-based assay and the TZMbl assay.<br/><br>
The glycans of Env can also take part in HIV host cell adhesion and infection, via host glycan-binding protein, such as galectins. To<br/><br>
explore this, we examined the interaction of gp120 (the surface Env protein) with a panel of galectins, by adapting the fluorescent<br/><br>
anisotropy (FA) assay to microscale. Galectin-8, a galectin with two carbohydrate recognition domains (CRDs), had high affinity for<br/><br>
gp120 as well as the HIV receptor CD4. The N-terminal CRD mediated the strongest interaction with gp120. The results of the FAassay<br/><br>
correlated well with binding of whole virions screened in another assay against the same panel of galectins, now immobilized on<br/><br>
beads. In the PR assay described above, here used as an infectivity assay, added intact galectin-8 enhanced infectivity of some HIV-1-<br/><br>
strains, while this was not seen with the N-CRD, demonstrating that both CRDs of galectin-8 are required for the effect on<br/><br>
infectivity. The enhancement effect mediated by galectin-8 was most pronounced with HIV-1 isolates obtained during the relative<br/><br>
immune competent chronic phase, as compared to viruses isolated after AIDS onset. Hence, galectin-8 binding carbohydrate motifs<br/><br>
on Env appear to be altered at severe immunodeficiency, adding to the knowledge on the evolution of Env glycosylation patterns<br/><br>
related to HIV pathogenesis. These results add to the basic knowledge of virus-host interactions, which hopefully could be used for<br/><br>
identification of antibodies and galectin-inhibitors effective in HIV prophylactic interventions.},
  author       = {Sheik-Khalil, Enas},
  isbn         = {978-91-7619-115-6},
  issn         = {1652-8220},
  keyword      = {HIV-1,galectin-1,galectin-8,neutralizing antibodies,cell-cell fusion,Microscale anisotropy assay,virus binding assay,glycosylation,gp120,Automated plaque reduction assay,CellProfiler,NeutNet},
  language     = {eng},
  pages        = {181},
  publisher    = {Microbiology, Immunology and Glycobiology, MIG, Lund University},
  school       = {Lund University},
  series       = {Lund University, Faculty of Medicine Doctoral Dissertation Series},
  title        = {Effects of galectins and antibodies in HIV infection; Novel Assays},
  volume       = {2015:36},
  year         = {2015},
}