Recent advances in the characterization of HIV-1 neutralization assays for standardized evaluation of the antibody response to infection and vaccination

Abstract

In AIDS vaccine development the pendulum has swung towards a renewed emphasis on the potential role for neutralizing antibodies in a successful global vaccine. It is recognized that vaccine-induced antibody performance, as assessed in the available neutralization assays, may well serve as a "gatekeeper" for HIV-1 subunit vaccine prioritization and advancement. As a result, development of a standardized platform for reproducible measurement of neutralizing antibodies has received considerable attention. Here we review current advancements in our knowledge of the performance of different types of antibodies in a traditional primary cell neutralization assay and the newer, more standardized TZM-bl reporter cell line assay. In light of recently revealed differences (see accompanying article) in the results obtained in these two neutralization formats, parallel evaluation with both platforms should be contemplated as an interim solution until a better understanding of immune correlates of protection is achieved.

Kinetic mechanism for HIV-1 neutralization by antibody 2G12 entails reversible glycan binding that slows cell entry

“…Indeed, it has been proposed that neutralization might ultimately require secondary processes such as NAb crosslinking, prevention of adsorption, or gp120 shedding (7)(8)(9)(10). A central cause for uncertainty occurs because the infectivity assays required to identify and analyze neutralization have been poorly understood and give discrepant results using identical HIV-1s and NAbs (11)(12)(13). Consequently, it has become evident that elucidation of NMAb mechanisms requires improved understanding of the factors that influence infectivity assays (11)(12)(13).…”

“…The most widely used neutralization assay, which has been recommended as a worldwide standard (13) and detects NAbs that contribute to host-virus coevolution in vivo (3,20), uses TZM-bl(JC.53) cells that were derived from our HeLa-CD4/ CCR5 cell clone JC.53 (21) by transfection with LTR-luciferase and LTR-β-galactosidase reporter plasmids (22). These reporters provide a facile means to monitor infections but have no effect on efficiencies of infection by diverse HIV-1 isolates or sensitivities to NAbs (23).…”

Designing a Soluble Near Full-length HIV-1 gp41 Trimer

“…Pseudovirus Neutralization Competition Assay-TZM/bl cells were used to determine HIV-1 neutralization by 2F5 and 4E10 mAbs (45,46). The mAb was titered in 3-fold serial dilutions starting at 50 g/ml in the growth medium (DMEM with 100 units/ml penicillin, 100 g/ml streptomycin, 2 mM L-glutamine (Quality Biologics Inc.), and 15% fetal calf serum (Gemini Bio-Products)).…”

“…IC 50 was calculated for each mAb alone and mAb pre-mixed with gp41 recombinant proteins or other control competitors. Two independent assays were performed, and the results were averaged (45,46).…”

“…Binding to 2F5 and 4E10 mAbs was determined by ELISA as described under "Experimental Procedures." C and D, virus neutralization was determined by the TZM/bl assay (45,46). Serial dilutions of purified 2F5 (C) or 4E10 (D) IgG were added to 96-well plates.…”