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. 2014 Oct:412:78-84.
doi: 10.1016/j.jim.2014.06.012. Epub 2014 Jun 19.

Improved flow-based method for HIV/SIV envelope-specific memory B-cell evaluation in rhesus macaques

Venkatramanan Mohanram  1 , Thorsten Demberg  1 , Iskra Tuero  1 , Diego Vargas-Inchaustegui  1 , George N Pavlakis  2 , Barbara K Felber  3 , Marjorie Robert-Guroff  4
Affiliations

Improved flow-based method for HIV/SIV envelope-specific memory B-cell evaluation in rhesus macaques

Venkatramanan Mohanram et al. J Immunol Methods. 2014 Oct.

Abstract

The ability to elicit potent and long-lasting broadly neutralizing HIV envelope (Env)-specific antibodies has become a key goal for HIV vaccine development. Consequently, the ability to rapidly and efficiently monitor development of memory B cells in pre-clinical and clinical vaccine trails is critical for continued progress in vaccine design. We have developed an improved flow cytometry-based method for the rapid and efficient identification of gp120-specific memory B cells in peripheral blood, bone marrow, and mucosal tissues which allows their direct staining without the need for prior cell sorting or enrichment. We demonstrate staining of both HIV and SIV Env-specific memory B cells in PBMC, bone marrow, and rectal tissue of vaccinated and infected rhesus macaques. Validation of the method is illustrated by statistically significant correlations with memory B cell levels quantified by ELISPOT assay and with serum binding antibody titers determined by ELISA. In addition to quantification, this method will bring the power of flow cytometry to the study of homing and trafficking of Env-specific memory B cells.

Keywords: Envelope-specific memory B cells; HIV; SIV.

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Figures

Figure 1
Figure 1. Gating strategy for identification of gp120-specific memory B cells in PBMC and rectal tissue
A) PBMC from a SHIV-challenged macaque were initially gated on a live/dead gate and CD2−, CD14− cells. This population was further gated for CD19+ and CD20+ cells to obtain a pure B cell population. IgD−, CD27+, gp120+ cells were considered as Env-specific memory B cells. The numbers in the plot indicate the frequency of the gated population. B) A representative plot comparing gp120-specific memory B cell staining in PBMC obtained at pre- and post-vaccinated time points from the same macaque. C) A single cell suspension from rectal tissue from a SHIV-challenged macaque was initially gated as in panel A. Following gating on CD19+CD20+ B cells, Env-specific memory B cells were identified as CD21+IgD−gp120+. D) A representative plot comparing gp120-specific memory B cell staining in rectal pinches obtained at pre- and post-vaccination time points from the same macaque.
Figure 2
Figure 2. Evaluation of gp120-specific memory B cells in SIV Env-vaccinated and HIV Env-vaccinated and SHIV-infected macaques
A) PBMC from Study 1 animals were assayed for SIV gp120-specific memory B cells prior to vaccination (Pre), two weeks after 2 vaccinations (Post-Prime), and two weeks after 4 vaccinations (Necropsy). B) PBMC from Study 2 animals were assayed for HIV gp120-specific memory B cells prior to vaccination (Pre), two weeks after the last immunization (Post-Protein boost), and two weeks after SHIV challenge (Post-Challenge). C) Bone marrow samples from Study 3 animals were assayed for SIV gp120-specific memory B cells prior to vaccination (Pre) and two weeks after the last immunization (Post-2nd boost). D) Rectal pinches from Study 3 macaques were quantified for SIV gp120-specific memory B cells two weeks after the last immunization (Post-protein boost), and 2 and 8 weeks Post-SIVmac251 challenge. Results for vector/adjuvant controls and vaccinated macaques (n = 36 are shown separately. Data presented are means ± SEM. p<0.05 indicates statistically significant differences between the indicated groups by the Mann-Whitney test.
Figure 3
Figure 3. gp120-specific memory B cells quantified by flow cytometry correlate with B cell ELISPOT data and antibody binding titers
(A, B) Correlation of SIV gp120-specific memory B cells in PBMC of Study 1 vaccinated macaques with bone marrow Env-specific memory B cells determined by ELISPOT, 2 weeks after two (A) and four (B) immunizations, respectively. (C, D) Correlation of HIV gp120-specific memory B cells in PBMC of Study 2 macaques with (C) Env-specific IgG- and (D) IgA-memory B cells in bone marrow determined by ELISPOT 2 weeks post-SHIV challenge. (E) Correlation of HIV gp120-specific memory B cells in PBMC of Study 2 vaccinated macaques with serum antibody titers determined by ELISA 2 weeks after the last immunization. (F) Lack of significant correlation of HIV gp120-specific memory B cells in PBMC of Study 2 SHIV-infected macaques 2 weeks post-infection with serum antibody titers obtained at the same time point. (G) Correlation of SIV gp120-specific memory B cells in bone marrow of Study 3 vaccinated macaques with matched bone marrow Env-specific memory B cells determined by ELISPOT, post-immunization. Correlation r and p values are from Spearman’s rank analysis. p<0.05 indicates statistically significant differences.
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References

    1. Bonsignori M, Moody MA, Parks RJ, Holl TM, Kelsoe G, Hicks CB, Vandergrift N, Tomaras GD, Haynes BF. HIV-1 envelope induces memory B cell responses that correlate with plasma antibody levels after envelope gp120 protein vaccination or HIV-1 infection. J Immunol. 2009;183(4):2708–2717. - PMC - PubMed
    1. Brocca-Cofano E, McKinnon K, Demberg T, Venzon D, Hidajat R, Xiao P, Daltabuit-Test M, Patterson LJ, Robert-Guroff M. Vaccine-elicited SIV and HIV envelope-specific IgA and IgG memory B cells in rhesus macaque peripheral blood correlate with functional antibody responses and reduced viremia. Vaccine. 2011;29(17):3310–3319. - PMC - PubMed
    1. Demberg T, Brocca-Cofano E, Kuate S, Aladi S, Vargas-Inchaustegui DA, Venzon D, Kalisz I, Kalyanaraman VS, Lee EM, Pal R, DiPasquale J, Ruprecht RM, Montefiori DC, Srivastava I, Barnett SW, Robert-Guroff M. Impact of antibody quality and anamnestic response on viremia control post-challenge in a combined Tat/Env vaccine regimen in rhesus macaques. Virology. 2013;440(2):210–221. - PMC - PubMed
    1. Demberg T, Brocca-Cofano E, Xiao P, Venzon D, Vargas-Inchaustegui D, Lee EM, Kalisz I, Kalyanaraman VS, Dipasquale J, McKinnon K, Robert-Guroff M. Dynamics of memory B-cell populations in blood, lymph nodes, and bone marrow during antiretroviral therapy and envelope boosting in simian immunodeficiency virus SIVmac251-infected rhesus macaques. J Virol. 2012;86(23):12591–12604. - PMC - PubMed
    1. Demberg T, Mohanram V, Venzon D, Robert-Guroff M. Phenotypes and distribution of mucosal memory B-cell populations in the SIV/SHIV rhesus macaque model. Clin Immunol. 2014 In press, 2014. http://dx.doi.org/10.1016/j.clim.2014.04.017. - DOI - PMC - PubMed

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