Abstract
The monitoring and assessment of a broadly neutralizing antibody (bnAb) based HIV-1 vaccine require detailed measurements of HIV-1 binding antibody responses to support the detection of correlates of protection. Here we describe the development of a flexible, high-throughput microsphere based multiplex assay system that allows monitoring complex binding antibody signatures. Studying a panel of 13 HIV-1 antigens in a parallel assessment of different IgG subclasses (IgG1, IgG2 and IgG3) we demonstrate the potential of our strategy. The technical advances we describe include means to improve antigen reactivity using directed neutravidin-biotin immobilization of antigens and biotin saturation to reduce background. A particular emphasis of our study was to provide tools for the assessment of reproducibility and stability of the assay system and strategies to control for variations allowing the application in high-throughput assays, where reliability of single measurements needs to be guaranteed.
Highlights
Neutralizing antibody responses are considered a key element of protective vaccines against HIV-1 (Burton and Hangartner, 2016)
The RV144 study used a canary pox vector (ALVAC-HIV vCP1521) to prime the immune system and recombinant gp120 AIDSVAX B/E boost while the study VAX003 primed with a single dose of recombinant gp120 AIDSVAX B/E, which emphasizes that the selection of antigen and regimen can influence the distribution of isotypes elicited during vaccination
We used the resurfaced core protein 3 (RSC3) (Wu et al, 2010), a protein designed to be preferentially recognized by CD4 binding site (CD4bs)-specific antibodies and the construct RSC3Δ (Wu et al, 2010), which lacks a crucial site required for CD4bs Ab binding, to assess the potentials of the assays
Summary
Neutralizing antibody (bnAb) responses are considered a key element of protective vaccines against HIV-1 (Burton and Hangartner, 2016). Evaluation of vaccine candidates in animal models and in human trials requires measuring of the desired function, a broad neutralization activity, and a thorough assessment of a broad range of binding antibody properties elicited by the vaccine to confirm correlates of protection This was highlighted by the RV144 study (Chung et al, 2014; Haynes et al, 2012), where non-neutralizing antibodies directed against the V2 region of the envelope protein were linked with the modest protection observed in this trial. The RV144 study used a canary pox vector (ALVAC-HIV vCP1521) to prime the immune system and recombinant gp120 AIDSVAX B/E boost while the study VAX003 primed with a single dose of recombinant gp120 AIDSVAX B/E, which emphasizes that the selection of antigen and regimen can influence the distribution of isotypes elicited during vaccination These observations highlight the importance of multiplex immunoassays to capture the complexity of antibody responses against HIV-1 and other pathogens
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