Gating strategy for plasmablast enumeration after hepatitis B vaccination

B cell stimulation develops upon vaccination, thus causing occurrence of activated B cells (plasmoblasts) in bloodstream. Similar cells are also observed in some viral infections. The contents of plasmablasts may be a marker of successful vaccination, or a diagnostic feature of ongoing infection. The plasmablasts are normally represented by a small cell subpopulation which is not easy to detect. A study was performed with 15 healthy volunteers who were subjected to a single immunization with a recombinant vaccine against hepatitis B virus. To identify the plasmablasts, we have used labeled antibodies prepared in our laboratory. These reagents were previously validated for counting the plasmablasts. Different gating strategies for plasmablast gating have been compared. Upon staining of lymphocytes from immunized volunteers, we observed a distinct cluster of plasmablasts with CD27⁺⁺CD38⁺⁺ phenotype using the following antibody set: CD19-PE, CD3/CD14/CD16-FITC, CD27-PC5.5 and CD38-PC7. Inclusion of a CD20-FITC antibody into the panel caused an increase of CD27⁺⁺CD38⁺⁺ plasmablast ratio among CD19⁺ lymphocytes to > 60%. Upon substitution of CD38 antibody by anti-CD71, a distinct plasmablast cluster was again revealed, which contained ca. 5 per cent В cells. Two strategies for the plasmablast gating using the CD27/ CD38 and CD27/CD71 combinations were compared in dynamics with lymphocyte samples from a single vaccinated volunteer. When applying the CD27/CD38 combination, a sharp and pronounced plasmablast peak was registered on day 7 post-vaccination. With CD27/CD71 combination, the peak was extended between day 7 and day 14 following immunization. Hence, time kinetics of the CD27⁺CD71⁺ population proved to be different from occurrence of classic plasmablasts with CD27⁺⁺CD38⁺⁺ phenotype. This finding suggests that the CD27⁺⁺CD71⁺population contains both plasmablasts and other types of activated B cells. A minor HBV surface antigen was prepared and labeled with phycoerythrin (HBsAg-PE), thus allowing to quantify the antigen-specific plasmablasts. The results of HBsAg-PE-based detection of antigen-specific cells were in compliance with the data obtained by ELISpot technique. At the present time, we use the original plasmablast gating technique for detection of activated B cells in SARS-CoV-2 infection. At the next step, this technique will be applied to sorting of antigen-specific B cells, thus permitting sequencing of Ig genes and design of novel human antibodies against viral antigens.

B cell stimulation develops upon vaccination, thus causing occurrence of activated B cells (plasmoblasts) in bloodstream. Similar cells are also observed in some viral infections. The contents of plasmablasts may be a marker of successful vaccination, or a diagnostic feature of ongoing infection. The plasmablasts are normally represented by a small cell subpopulation which is not easy to detect. A study was performed with 15 healthy volunteers who were subjected to a single immunization with a recombinant vaccine against hepatitis B virus. To identify the plasmablasts, we have used labeled antibodies prepared in our laboratory. These reagents were previously validated for counting the plasmablasts. Different gating strategies for plasmablast gating have been compared. Upon staining of lymphocytes from immunized volunteers, we observed a distinct cluster of plasmablasts with CD27⁺⁺CD38⁺⁺ phenotype using the following antibody set: CD19-PE, CD3/CD14/CD16-FITC, CD27-PC5.5 and CD38-PC7. Inclusion of a CD20-FITC antibody into the panel caused an increase of CD27⁺⁺CD38⁺⁺ plasmablast ratio among CD19⁺ lymphocytes to > 60%. Upon substitution of CD38 antibody by anti-CD71, a distinct plasmablast cluster was again revealed, which contained ca. 5 per cent В cells. Two strategies for the plasmablast gating using the CD27/ CD38 and CD27/CD71 combinations were compared in dynamics with lymphocyte samples from a single vaccinated volunteer. When applying the CD27/CD38 combination, a sharp and pronounced plasmablast peak was registered on day 7 post-vaccination. With CD27/CD71 combination, the peak was extended between day 7 and day 14 following immunization. Hence, time kinetics of the CD27⁺CD71⁺ population proved to be different from occurrence of classic plasmablasts with CD27⁺⁺CD38⁺⁺ phenotype. This finding suggests that the CD27⁺⁺CD71⁺ population contains both plasmablasts and other types of activated B cells. A minor HBV surface antigen was prepared and labeled with phycoerythrin (HBsAg-PE), thus allowing to quantify the antigen-specific plasmablasts. The results of HBsAg-PE-based detection of antigen-specific cells were in compliance with the data obtained by ELISpot technique. At the present time, we use the original plasmablast gating technique for detection of activated B cells in SARS-CoV-2 infection. At the next step, this technique will be applied to sorting of antigen-specific B cells, thus permitting sequencing of Ig genes and design of novel human antibodies against viral antigens.

1. Budkova A.I., Lapin S.V., Serebriakova M.K., Kudryavtsev I.V., Trishina I.N., Maslyansky A.L., Totolian Areg A. B-cell subpopulations of peripheral blood in systemic lupus erythematosus. Meditsinskaya immunologiya = Medical Immunology (Russia), 2017, Vol. 19, no. 2, pp. 175-184. (In Russ.) doi: 10.15789/1563-0625-2017-2-175-184.

2. Kudryavtsev I.V., Subbotovskaya A.I. Application of six-color flow cytometric analysis for immune profile monitoring. Meditsinskaya immunologiya = Medical Immunology (Russia), 2015, Vol. 17, no. 1, pp. 19-26. (In Russ.) doi: 10.15789/1563-0625-2015-1-19-26.

3. Khaydukov S., Baidun L., Zurochka A., Totolyan Areg A. Methods. Meditsinskaya immunologiya = Medical Immunology (Russia), 2012, Vol. 14, no. 3, pp. 255-268. (In Russ.) doi: 10.15789/1563-0625-2012-3-255-268.

4. Amanna I.J., Slifka M.K. Quantitation of rare memory B cell populations by two independent and complementary approaches. J. Immunol. Methods, 2006, Vol. 317, pp. 175-185.

5. Basha S., Pichichero M.E. Poor memory B cell generation contributes to non-protective responses to DTaP vaccine antigens in otitis-prone children. Clin. Exp. Immunol., 2015, Vol. 182, no. 3, pp. 314-322.

6. Bauer T., Jilg W. Hepatitis B surface antigen-specific T and B cell memory in individuals who had lost protective antibodies after hepatitis B vaccination. Vaccine, 2006, Vol. 24, pp. 572-577.

7. Berg E.A., Fishman J.B. Labeling antibodies with Cy5-Phycoerythrin. Cold Spring Harb. Protoc., 2019, Vol. 9, 099317. https://doi.org/10.1101/pdb.prot099317.

8. Buisman A.M., de Rond C.G., Ozturk K., Ten Hulscher H.I., van Binnendijk R.S. Long-term presence of memory B-cells specific for different vaccine components. Vaccine, 2009, Vol. 28, pp. 179-186.

9. Cao Y., Gordic M., Kobold S., Lajmi N., Meyer S., Bartels K., Hildebrandt Y., Luetkens T., Ihloff A.S., Kroger N., Bokemeyer C., Atanackovic D. An optimized assay for the enumeration of antigen-specific memory B cells in different compartments of the human body. J. Immunol. Methods, 2010, Vol. 358, no. 1-2, pp. 56-65.

10. Covens K., Verbinnen B., Geukens N., Meyts I., Schuit F., Van Lommel L., Jacquemin M., Bossuyt X. Characterization of proposed human B-1 cells reveals pre-plasmablast phenotype. Blood, 2013, Vol. 121, no. 26, pp. 5176-5183.

11. Ellebedy A.H., Jackson K.J., Kissick H.T., Nakaya H.I., Davis C.W., Roskin K.M., McElroy A.K., Oshansky C.M., Elbein R., Thomas S., Lyon G.M., Spiropoulou C.F., Mehta A.K., Thomas P.G., Boyd S.D., Ahmed R. Defining antigen-specific plasmablast and memory B cell subsets in blood following viral infection and vaccination of humans. Nat. Immunol., 2016, Vol. 17, no. 10, pp. 1226-1234.

12. Engel P., Boumsell L., Balderas R., Bensussan A., Gattei V., Horejsi V., Jin B.Q., Malavasi F., Mortari F., Schwartz-Albiez R., Stockinger H., van Zelm M.C., Zola H., Clark G. CD Nomenclature 2015: human leukocyte differentiation antigen workshops as a driving force in immunology. J. Immunol., 2015, Vol. 195, no. 10, pp. 4555-4563.

13. Fink K. Origin and function of circulating plasmablasts during acute viral infections. Front. Immunol., 2012, Vol. 3, no. 78, pp. 1-5.

14. Frölich D., Giesecke C., Mei H.E., Reiter K., Daridon C., Lipsky P.E., Dörner T. Secondary immunization generates clonally related antigen-specific plasma cells and memory B cells. J. Immunol., 2010, Vol. 185, no. 5, pp. 3103-3110.

15. Galson J.D., Pollard A.J., Trück J., Kelly D.F. Studying the antibody repertoire after vaccination: practical applications. Trends Immunol., 2014, Vol. 35, no. 7, pp. 319-331.

16. Georgiou G., Ippolito G.C., Beausang J., Busse C.E., Wardemann H., Quake S.R. The promise and challenge of high-throughput sequencing of the antibody repertoire. Nat. Biotechnol., 2014, Vol. 32, no. 2, pp. 158-168.

17. Kaminski D.A., Wei C., Qian Y., Rosenberg A.F., Sanz I. Advances in human B cell phenotypic profiling. Front. Immunol., 2012, Vol. 3, 302. doi: 10.3389/fimmu.2012.00302.

18. Khvastunova A.N., Kuznetsova S.A., Al-Radi L.S., Vylegzhanina A.V., Zakirova A.O., Fedyanina O.S., Filatov A.V., Vorobjev I.A., Ataullakhanov F. Anti-CD antibody microarray for human leukocyte morphology examination allows analyzing rare cell populations and suggesting preliminary diagnosis in leukemia. Sci. Rep., 2015, Vol. 5, no. 12573, pp. 1-13.

19. Kuri-Cervantes L., Pampena M.B., Meng W., Rosenfeld A.M., Ittner C.A.G., Weisman A.R., Agyekum R., Mathew D., Amy E., Baxter A.E., Vella L., Kuthuru O., Apostolidis S., Bershaw L., Dougherty J., Greenplate A.R., Pattekar A., Kim J., Han N., Gouma S., Weirick M.E., Arevalo C.P., Bolton M.J., Goodwin E.C., Anderson E.M., Hensley S.E., Jones T.K., Mangalmurti N.S., Prak E.T.L., Wherry E.J., Meyer N.J., Betts M.R. Immunologic perturbations in severe COVID-19/SARS-CoV-2 infection. bioRxiv, 2020, 101717. doi: 10.1101/2020.05.18.101717.

20. Lee F.E., Falsey A.R., Halliley J.L., Sanz I., Walsh E.E. Circulating antibody-secreting cells during acute respiratory syncytial virus infection in adults. J. Infect. Dis., 2010, Vol. 202, no. 11, pp. 1659-1666.

21. Leggat D.J., Khaskhely N.M., Iyer A.S., Mosakowski J., Thompson R.S., Weinandy J.D., Westerink M.A. Pneumococcal polysaccharide vaccination induces polysaccharide-specific B cells in adult peripheral blood expressing CD19+CD20+CD3- CD70- CD27+IgM+CD43+CD5+/- . Vaccine, 2013, Vol. 31, pp. 4632-4640.

22. Lin Z., Chiang N.Y., Chai N., Seshasayee D., Lee W.P., Balazs M., Nakamura G., Swem L.R. In vivo antigendriven plasmablast enrichment in combination with antigen-specific cell sorting to facilitate the isolation of rare monoclonal antibodies from human B cells. Nat. Protoc., 2014, Vol. 9, no. 7, pp. 1563-1577.

23. Lu D.R., Tan Y.C., Kongpachith S., Cai X., Stein E.A., Lindstrom T.M., Sokolove J., Robinson W.H. Identifying functional anti-Staphylococcus aureus antibodies by sequencing antibody repertoires of patient plasmablasts. Clin. Immunol., 2014, Vol. 152, no. 1-2, pp. 77-89.

24. Mahnke Y.D., Roederer M. Optimizing a multicolor immunophenotyping assay. Clin. Lab. Med., 2007, Vol. 27, pp. 469-485.

25. Meyer J.P., Adumeau P., Lewis J.S., Zeglis B.M. Click сhemistry and radiochemistry: the first 10 years. Bioconjug. Chem., 2016, Vol. 27, no. 12, pp. 2791-2807.

26. Nakamura G., Chai N., Park S., Chiang N., Lin Z., Chiu H., Fong R., Yan D., Kim J., Zhang J., Lee W.P., Estevez A., Coons M., Xu M., Lupardus P., Balazs M., Swem L.R. An in vivo human-plasmablast enrichment technique allows rapid identification of therapeutic influenza A antibodies. Cell Host Microbe, 2013, Vol. 14, no. 1, pp. 93-103.

27. Pallikkuth S., Pilakka Kanthikeel S., Silva S.Y., Fischl M., Pahwa R., Pahwa S. Upregulation of IL-21 receptor on B cells and IL-21 secretion distinguishes novel 2009 H1N1 vaccine responders from nonresponders among HIVinfected persons on combination antiretroviral therapy. J. Immunol., 2011, Vol. 186, no. 11, pp. 6173-6281.

28. Plotkin S.A. Correlates of protection induced by vaccination. Clin. Vaccine Immunol., 2010, Vol. 17, no. 7, pp. 1055-1065.

29. Robinson W.H. Sequencing the functional antibody repertoire – diagnostic and therapeutic discovery. Nat. Rev. Rheumatol., 2015, Vol. 11, no. 3, pp. 171-182.

30. Roederer M. Spectral compensation for flow cytometry: visualization artifacts, limitations, and caveats. Cytometry, 2001, Vol. 45, no. 3, pp. 194-205.

31. Simons B.C., Spradling P.R., Bruden D.J.T., Zanis C., Case S., Choromanski T.L., Apodaca M., Brogdon H.D., Dwyer G., Snowball M., Negus S., Bruce M.G., Morishima C., Knall C., McMahon B.J. A longitudinal hepatitis B vaccine cohort demonstrates long-lasting hepatitis B virus (HBV) cellular immunity despite loss of antibody against HBV surface antigen. J. Infect., 2016, Vol. 214, no. 2, pp. 273-280.

32. Smith K., Crowe S.R., Garman L., Guthridge C.J., Muther J.J., McKee E., Zheng N.Y., Farris A.D., Guthridge J.M., Wilson P.C., James J.A. Human monoclonal antibodies generated following vaccination with AVA provide neutralization by blocking furin cleavage but not by preventing oligomerization. Vaccine, 2012, Vol. 30, no. 28, pp. 4276-4283.

33. Smith K., Garman L., Wrammert J., Zheng N.Y., Capra J.D., Ahmed R., Wilson P.C. Rapid generation of fully human monoclonal antibodies specific to a vaccinating antigen. Nat. Protoc., 2009, Vol. 4, no. 3, pp. 372-384.

34. Stathopoulos P., Kumar A., Nowak R.J., O’Connor K.C. Autoantibody-producing plasmablasts after B cell depletion identified in muscle-specific kinase myasthenia gravis. JCI Insight, 2017, Vol. 2, no. 17, e94263. doi: 10.1172/jci.insight.94263.

35. Tian C., Chen Y., Liu Y., Wang S., Li Y., Wang G., Xia J., Zhao X. A., Huang R., Lu S., Wu C. Use of ELISpot assay to study HBs-specific B cell responses in vaccinated and HBV infected humans. Emerg. Microbes Infect., 2018, Vol. 7, no. 1, 16. doi: 10.1038/s41426-018-0034-0.

36. Vira S., Mekhedov E., Humphrey G., Blank P.S. Fluorescent-labeled antibodies: balancing functionality and degree of labeling. Anal. Biochem., 2010, Vol. 402, no. 2, pp. 146-150.

37. Wilson P.C., Andrews S.F. Tools to therapeutically harness the human antibody response. Nat. Rev. Immunol., 2012, Vol. 12, no. 10, pp. 709-719.