International network for comparison of HIV neutralization assays: the NeutNet report

BACKGROUND: Neutralizing antibody assessments play a central role in human immunodeficiency virus type-1 (HIV-1) vaccine development but it is unclear which assay, or combination of assays, will provide reliable measures of correlates of protection. To address this, an international collaboration (NeutNet) involving 18 independent participants was organized to compare different assays. METHODS: Each laboratory evaluated four neutralizing reagents (TriMab, 447-52D, 4E10, sCD4) at a given range of concentrations against a panel of 11 viruses representing a wide range of genetic subtypes and phenotypes. A total of 16 different assays were compared. The assays utilized either uncloned virus produced in peripheral blood mononuclear cells (PBMCs) (virus infectivity assays, VI assays), or their Env-pseudotyped (gp160) derivatives produced in 293T cells (PSV assays) from molecular clones or uncloned virus. Target cells included PBMC and genetically-engineered cell lines in either a single- or multiple-cycle infection format. Infection was quantified by using a range of assay read-outs that included extracellular or intracellular p24 antigen detection, RNA quantification and luciferase and beta-galactosidase reporter gene expression. FINDINGS: PSV assays were generally more sensitive than VI assays, but there were important differences according to the virus and inhibitor used. For example, for TriMab, the mean IC50 was always lower in PSV than in VI assays. However, with 4E10 or sCD4 some viruses were neutralized with a lower IC50 in VI assays than in the PSV assays. Inter-laboratory concordance was slightly better for PSV than for VI assays with some viruses, but for other viruses agreement between laboratories was limited and depended on both the virus and the neutralizing reagent. CONCLUSIONS: The NeutNet project demonstrated clear differences in assay sensitivity that were dependent on both the neutralizing reagent and the virus. No single assay was capable of detecting the entire spectrum of neutralizing activities. Since it is not known which in vitro assay correlates with in vivo protection, a range of neutralization assays is recommended for vaccine evaluation.The project “NeutNet: Standardisation of HIV neutralisation assays to be used in vaccine research and clinical trials” was sponsored by EC grant number LSSP-CT-2004-012190. The WHO/UNAIDS HIV Vaccine Initiative provided partial support for the conduct of the project, including the activities of the Repository, preparation and shipment of reagents. Additional support was received from EUROPRISE Network of Excellence of the EU, grant number LSHP CT-2006-037611; MS-Brasil PN DST/AIDS 188/04; Instituto de Salud Carlos III (RETIC RD06/0006) and FIPSE Foundation (36536/05); the Fund for Scientific Research in Belgium (FWO-Vlaanderen, grant number G.0346.03); the Istituto Superiore di Sanità -VI Programma Nazionale (grant numbers 45G.35 and 40G.56); the Swedish Research Council and the Swedish International Development Cooperation Agency/Department for Research Cooperation (SIDA/SAREC); The Bill and Melinda Gates Foundation Collaboration for AIDS vaccine discovery (CAVD) and the International AIDS Vaccine Initiative (IAVI) Neutralizing Antibody Consortium (NAC). Antibody 447-52D was prepared through the auspices of the NIH-sponsored New York University Center for AIDS Research supported by grant AI 27742. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.S