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. 2009 Jan 1;340(1):42-7.
doi: 10.1016/j.jim.2008.09.025. Epub 2008 Nov 6.

Frequencies of human influenza-specific antibody secreting cells or plasmablasts post vaccination from fresh and frozen peripheral blood mononuclear cells

Shuya Y Kyu  1 , James Kobie, Hongmei Yang, Martin S Zand, David J Topham, Sally A Quataert, Ignacio Sanz, F Eun-Hyung Lee
Affiliations

Frequencies of human influenza-specific antibody secreting cells or plasmablasts post vaccination from fresh and frozen peripheral blood mononuclear cells

Shuya Y Kyu et al. J Immunol Methods. .

Abstract

The rise in influenza-specific neutralizing antibody levels is proceeded by a burst of antigen-specific antibody secreting cells (ASC) or plasmablasts identified in peripheral blood approximately 5-10 days post immunization. Blood antigen-specific ASC may function as an immune marker of vaccine responses in comparison to the pre- and post-neutralizing titers; however, some have questioned whether there is adequate survival of ASC isolated from peripheral blood after freezing, making multi-center vaccine trials difficult. Here, we demonstrate similar frequencies of influenza-specific ASC from fresh and frozen peripheral blood mononuclear cells (PBMC). Influenza Hemagglutinin (HA) H1, H3, and H7-specific ASC IgG ELISpots frequencies were compared from the same fresh and frozen PBMC 7 days after 2006 Trivalent Influenza Vaccine (TIV) in 10 young healthy subjects. H1-, H3-, and H7-specific IgG ASC spots/10(6) from fresh PBMC on day 7 were 229+/-341, 98+/-90, and 6+/-11 respectively. Total IgG ASC spots/million PBMC pre- and 7-day post-vaccination were 290+/-188 (0.029% PBMC) and 1691+/-836 (0.17% PBMC) respectively. There was no difference in the H1 -H3-, and total specific ASC IgG ELISpot frequencies from the fresh versus frozen PBMC on day 7 (p=0.43, 0.28, 0.28 respectively). These results demonstrate feasibility of testing whether antigen-specific ASC from frozen PBMC are an early biomarker of long-term antibody responses in multi-center vaccine trials.

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Figures

Figure 1
Figure 1
Comparison of influenza-specific and total IgG ASC from fresh PBMC of 10 subjects pre and 7 day-post TIV immunization. All spots shown had background wells subtracted. No H1- or H3-specific ASC spots/106 PBMC are noted on day 0. Increased H1- (green, top left) and H3-specific (blue, top right) ASC spots/106 PBMC were seen on day 7 compared to day 0 (Wilcoxian Rank Test, p= 0.002 and p= 0.006 respectively). Minimal avian H7 responses (red, bottom right) were noted on 7-day post vaccination (p= 0.06) and functioned as a negative control. A rise in total IgG (black, bottom right) ASC were seen in the blood from day 0 to 7 (p=0.002)
Figure 2
Figure 2
Comparison of influenza-specific and total IgG ASC frequencies 7-days post TIV vaccine from the same fresh and frozen PBMC sample. All spots shown had background wells subtracted. There was no difference in the fresh and frozen samples for H1- (top left) and H3-(top right) specific ASC spots/106 PBMC from fresh (H1-green, H3-blue) and frozen (corresponding gray) PBMC (Wilcoxian Rank Test, p= 0.43, p=0.28, respectively). Fresh and frozen H7 (bottom right) ASC spots/106 PBMC were minimal and similar. The total IgG (bottom right) ASC spots/106 PBMC were also not different from the fresh and frozen samples (p=0.28).
Figure 3
Figure 3
Comparison of plasmablasts or ASC by flow cytometry from the same fresh and frozen PBMC samples of 3 subjects are shown. Plasmablasts were identified as CD19+ CD27hi and CD28hi. Total PBMC numbers were identified by FSC and SSC. Numbers of ASC by flow cytometry are presented per 106 PBMC.
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