Effects of galectins and antibodies in HIV infection; Novel Assays

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

Rational design of HIV vaccines and microbicides: report of the EUROPRISE network annual conference 2010

Novel, exciting intervention strategies to prevent infection with HIV have been tested in the past year, and the field is rapidly evolving. EUROPRISE is a network of excellence sponsored by the European Commission and concerned with a wide range of activities including integrated developmental research on HIV vaccines and microbicides from discovery to early clinical trials. A central and timely theme of the network is the development of the unique concept of co-usage of vaccines and microbicides. This review, prepared by the PhD students of the network captures much of the research ongoing between the partners. The network is in its 5th year and involves over 50 institutions from 13 European countries together with 3 industrial partners; GSK, Novartis and Sanofi-Pasteur. EUROPRISE is involved in 31 separate world-wide trials of Vaccines and Microbicides including 6 in African countries (Tanzania, Mozambique, South Africa, Kenya, Malawi, Rwanda), and is directly supporting clinical trials including MABGEL, a gp140-hsp70 conjugate trial and HIVIS, vaccine trials in Europe and Africa

International Network for Comparison of HIV Neutralization Assays: The NeutNet Report II

BACKGROUND: Neutralizing antibodies provide markers for vaccine-induced protective immunity in many viral infections. By analogy, HIV-1 neutralizing antibodies induced by immunization may well predict vaccine effectiveness. Assessment of neutralizing antibodies is therefore of primary importance, but is hampered by the fact that we do not know which assay(s) can provide measures of protective immunity. An international collaboration (NeutNet) involving 18 different laboratories previously compared different assays using monoclonal antibodies (mAbs) and soluble CD4 (Phase I study). METHODS: In the present study (Phase II), polyclonal reagents were evaluated by 13 laboratories. Each laboratory evaluated nine plasmas against an 8 virus panel representing different genetic subtypes and phenotypes. TriMab, a mixture of three mAbs, was used as a positive control allowing comparison of the results with Phase I in a total of nine different assays. The assays used either uncloned virus produced in peripheral blood mononuclear cells (PBMCs) (Virus Infectivity Assays, VIA), or Env (gp160)-pseudotyped viruses (pseudoviruses, PSV) produced in HEK293T cells from molecular clones or from uncloned virus. Target cells included PBMC and genetically engineered cell lines in either single- or multiple-cycle infection format. Infection was quantified by using a range of assay read-outs including extra- or intra-cellular p24 antigen detection, luciferase, beta-galactosidase or green fluorescent protein (GFP) reporter gene expression. FINDINGS: Using TriMab, results of Phase I and Phase II were generally in agreement for six of the eight viruses tested and confirmed that the PSV assay is more sensitive than PBMC (p = 0.014). Comparisons with the polyclonal reagents showed that sensitivities were dependent on both virus and plasma. CONCLUSIONS: Here we further demonstrate clear differences in assay sensitivities that were dependent on both the neutralizing reagent and the virus. Consistent with the Phase I study, we recommend parallel use of PSV and VIA for vaccine evaluation

Generation of neutralizing antibodies and divergence of SIVmac239 in cynomolgus macaques following short-term early antiretroviral therapy.

Neutralizing antibodies (NAb) able to react to heterologous viruses are generated during natural HIV-1 infection in some individuals. Further knowledge is required in order to understand the factors contributing to induction of cross-reactive NAb responses. Here a well-established model of experimental pathogenic infection in cynomolgus macaques, which reproduces long-lasting HIV-1 infection, was used to study the NAb response as well as the viral evolution of the highly neutralization-resistant SIVmac239. Twelve animals were infected intravenously with SIVmac239. Antiretroviral therapy (ART) was initiated ten days post-inoculation and administered daily for four months. Viral load, CD4(+) T-cell counts, total IgG levels, and breadth as well as strength of NAb in plasma were compared simultaneously over 14 months. In addition, envs from plasma samples were sequenced at three time points in all animals in order to assess viral evolution. We report here that seven of the 12 animals controlled viremia to below 10(4) copies/ml of plasma after discontinuation of ART and that this control was associated with a low level of evolutionary divergence. Macaques that controlled viral load developed broader NAb responses early on. Furthermore, escape mutations, such as V67M and R751G, were identified in virus sequenced from all animals with uncontrolled viremia. Bayesian estimation of ancestral population genetic diversity (PGD) showed an increase in this value in non-controlling or transient-controlling animals during the first 5.5 months of infection, in contrast to virus-controlling animals. Similarly, non- or transient controllers displayed more positively-selected amino-acid substitutions. An early increase in PGD, resulting in the generation of positively-selected amino-acid substitutions, greater divergence and relative high viral load after ART withdrawal, may have contributed to the generation of potent NAb in several animals after SIVmac239 infection. However, early broad NAb responses correlated with relatively preserved CD4(+) T-cell numbers, low viral load and limited viral divergence

Labcode 9B - HIV neutralization Protocol automated Plaque Reduction Assay with GHOST cells

This document contains a published paper with a detailed protocol on a high-throughput, high-content automated plaque reduction (APR) assay based on automated microscopy and image analysis that allows evaluation of neutralization and inhibition of cell-cell fusion within the same assay. Neutralization of virus particles is measured as a reduction in the number of fluorescent plaques, and inhibition of cell-cell fusion as a reduction in plaque area. PMID: 2517603

Automated image-based assay for evaluation of HIV neutralization and cell-to-cell fusion inhibition

Background: Standardized techniques to detect HIV-neutralizing antibody responses are of great importance in the search for an HIV vaccine. Methods: Here, we present a high-throughput, high-content automated plaque reduction (APR) assay based on automated microscopy and image analysis that allows evaluation of neutralization and inhibition of cell-cell fusion within the same assay. Neutralization of virus particles is measured as a reduction in the number of fluorescent plaques, and inhibition of cell-cell fusion as a reduction in plaque area. Results: We found neutralization strength to be a significant factor in the ability of virus to form syncytia. Further, we introduce the inhibitory concentration of plaque area reduction (ICpar) as an additional measure of antiviral activity, i.e. fusion inhibition. Conclusions: We present an automated image based high-throughput, high-content HIV plaque reduction assay. This allows, for the first time, simultaneous evaluation of neutralization and inhibition of cell-cell fusion within the same assay, by quantifying the reduction in number of plaques and mean plaque area, respectively. Inhibition of cell-to-cell fusion requires higher quantities of inhibitory reagent than inhibition of virus neutralization

Generation of neutralizing antibodies and divergence of SIVmac239 in cynomolgus macaques following short-term early antiretroviral therapy

Neutralizing antibodies (NAb) able to react to heterologous viruses are generated during natural HIV-1 infection in some individuals. Further knowledge is required in order to understand the factors contributing to induction of cross-reactive NAb responses. Here a well-established model of experimental pathogenic infection in cynomolgus macaques, which reproduces long-lasting HIV-1 infection, was used to study the NAb response as well as the viral evolution of the highly neutralization-resistant SIVmac239. Twelve animals were infected intravenously with SIVmac239. Antiretroviral therapy (ART) was initiated ten days post-inoculation and administered daily for four months. Viral load, CD4+ T-cell counts, total IgG levels, and breadth as well as strength of NAb in plasma were compared simultaneously over 14 months. In addition, envs from plasma samples were sequenced at three time points in all animals in order to assess viral evolution. We report here that seven of the 12 animals controlled viremia to below 104 copies/ml of plasma after discontinuation of ART and that this control was associated with a low level of evolutionary divergence. Macaques that controlled viral load developed broader NAb responses early on. Furthermore, escape mutations, such as V67M and R751G, were identified in virus sequenced from all animals with uncontrolled viremia. Bayesian estimation of ancestral population genetic diversity (PGD) showed an increase in this value in non-controlling or transient-controlling animals during the first 5.5 months of infection, in contrast to virus-controlling animals. Similarly, non- or transient controllers displayed more positively-selected amino-acid substitutions. An early increase in PGD, resulting in the generation of positively-selected amino-acid substitutions, greater divergence and relative high viral load after ART withdrawal, may have contributed to the generation of potent NAb in several animals after SIVmac239 infection. However, early broad NAb responses correlated with relatively preserved CD4+ T-cell numbers, low viral load and limited viral divergence

Comparison of PSV and VI assays across viruses.

<p>(A) circular “radar” plots. Lines from the centre represent an axis for each virus. The geometric mean IC value for PSV (blue lines) and PBMC (red lines) against each virus is plotted, and the points joined. The scale is set such that the centre represents no neutralization and the concentric grid-lines are 2-fold dilution steps moving out to highest neutralization at the edge. (B) and (C) Ranking of viruses for relative sensitivity to neutralization was done by calculating geometric mean IC50s across laboratories (grouping PSV and PBMC separately). (B) Ranking by TriMab and (C) ranking by plasma (means over ARP515-522). The scale is set such that the most neutralization sensitive viruses are at the top.</p

Neutralization assays and their characteristics.

<p>Cell target: PBMC, peripheral blood mononuclear cells; the cell lines GHOST, U87 and HeLa are stably transfected with CD4 and CCR5 or CXCR4. MR, multiple round infection; SR, single round infection. The fusion assay is limited to cell surface-viral envelope interaction. Ab persistence: time of incubation of the inhibitory reagent with virus and cells before washout. Day: time of read-out, numbers indicate days; hr, hours. Env plasmid, Env expression plasmids obtained through NIBSC.</p