Interview with the author: HIV-1 neutralization assay

Mitigation of variation observed in a peripheral blood mononuclear cell (PBMC) based HIV-1 neutralization assay by donor cell pooling

Read the full article on ScienceDirect.

Interview with first author Lindsay Wieczorek

What problem does this method address?

In this study, we pool peripheral blood mononuclear cells (PBMC) from healthy donors to use as assay targets for HIV neutralization assays. Assessing antibody-mediated neutralization in the HIV’s natural host cells may best represent physiologic events that occur during HIV infection. However, the PBMC neutralization assay is plagued with variability, introduced by normal donor variation, making it difficult to compare neutralization assay results generated using different individual PBMC.  To mitigate the influence of individual donors on the assay endpoint, we pool PBMC from multiple donors to achieve an ‘average’ response. This approach helps improve assay standardization.

How did you develop it?

Other groups, including ours, have previously used pooled PBMC for neutralization assay targets. However, the effect of donor variation on assay endpoints and the use of pooling PBMC to reduce variation had not been fully characterized or proposed for use in vaccine studies. We systematically addressed these issues and better defined donor influence on neutralizing antibody titers. In addition, we used infectious molecular clones (IMC) containing the luciferase reporter gene, developed at the University of Alabama (Edmonds, et al., Virology 408:1-13, 2010), and at our institute (Chenine, et al., PLoS ONE, in press) to improve assay throughput and endpoint detection.

How does it improve on existing methods?

Pooling of PBMC from multiple donors reduces variation in neutralization assay endpoint compared to use of different individual PBMC for assay targets.  Using multiple cryopreserved leukopaks to create pools generates a more sustainable PBMC source, useful for analysis of large clinical trials. We use reporter IMC to facilitate assay readout; endpoint is detected as relative light units, and since endpoint doesn’t require quantitation of viral protein, the antibody and the viral inoculum can remain in culture for the duration of the assay. This allows the antibody to function through multiple rounds of HIV replication and provides a more physiologic approach.

What tips and tricks do you have for using this method?

Effective cryopreservation with high cell viability is critical. We pre-screen the donor cells for HIV replication to avoid including non-permissive donors, which would result in poor infectivity and higher assay variability. We exclude donors who are positive for the CCR5-Δ32 polymorphism, as those HIV-seronegative donors are very non-permissive for HIV replication. If flow cytometry phenotyping is possible, we recommend use of donors with higher CD4+ cells and lower NK cells for achieving higher virus replication in the cultures. Additionally, individual PBMC need to be stimulated separately then pooled right before performing the neutralization assay to avoid a mixed lymphocyte reaction.

Introducing the author
Lindsay Wieczorek – Laboratory of Vaccine Immunology, U.S. Military HIV Research Program (MHRP), HJF

About the research

Mitigation of variation observed in a peripheral blood mononuclear cell (PBMC) based HIV-1 neutralization assay by donor cell pooling
Lindsay Wieczorek, Bruce K. Brown, Camila DelSarto Macedo, Maggie Wesberry-Schmierer, Viseth Ngauy, Andrew Rosa Borges, Nelson L. Michael, Mary A. Marovich, David C. Montefiori, Victoria R. Polonis
Virology, Volume 447, Issues 1–2, December 2013, Pages 240–248

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