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.Funding: The project “NeutNet: Standardisation of HIV neutralization assays to be used in vaccine research and clinical trials” was sponsored by the European Community under grant numbers LSSP-CT-2004-012190, EUROPRISE-Network of Excellence grant number LSHP CT-2006-037611 and NGIN grant number 201433. The WHO/UNAIDS HIV Vaccine Initiative provided partial support for the conduct of the project, including the activities of the Repository, such as preparation and shipment of reagents. Additional support was received from The Bill and Melinda Gates Foundation Collaboration for AIDS Vaccine Discovery (CAVD), and Departamento de DST, Aids e Hepatites Virais, MS-Brasil 147/08. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.S