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. 1995 Aug;69(8):4888–4897. doi: 10.1128/jvi.69.8.4888-4897.1995

Selection of a single amino acid substitution in the hemagglutinin molecule by chicken eggs can render influenza A virus (H3) candidate vaccine ineffective.

S Kodihalli 1, D M Justewicz 1, L V Gubareva 1, R G Webster 1
PMCID: PMC189303  PMID: 7609057

Abstract

This study investigated whether a single amino acid change in the hemagglutinin (HA) molecule influenced the efficacy of formalin-inactivated influenza A (H3N1) vaccine candidates derived from high-growth reassortants between the standard donor of high-yield genes (A/PR/8/34 [H1N1]) and host cell variants generated from the same clinical isolate (A/Memphis/7/90 [H3N2]) by passage in embryonated chicken eggs. Two clones of the isolate generated by growth in eggs differed from the parent virus (represented by an MDCK cell-grown counterpart) solely by the presence of Lys (instead of Glu) at position 156 or Ile (instead of Ser) at position 186 in the HA1 subunit. The protective efficacy of egg-grown HA Lys-156 and HA Ile-186 reassortant variants was compared with that of the MDCK cell-grown reassortant vaccine. Classically, antibody titers in serum have been used to demonstrate vaccine efficacy. Here, parameters of B-cell responsiveness were monitored, including the kinetics, character, and localization of the primary antibody-forming cell (AFC) response and the development of B-cell memory in lymphoid tissues associated with the priming site (spleen) and responsive to pulmonary challenge with infectious virus (upper and lower respiratory tract lymph nodes). We show that the egg-grown HA Lys-156 variant induced an AFC profile vastly different from that elicited by the other two reassortant vaccines. The vaccine was poorly immunogenic; it induced antibodies that were cross-reactive prior to challenge but which, postchallenge with a lethal dose of the MDCK cell-grown reassortant virus, were targeted primarily to the HA Lys-156 variant, were of the immunoglobulin M isotype, were nonprotective, and were derived from the spleen. In contrast, the egg-grown HA Ile-186 variant was remarkably like the MDCK cell-grown virus in that protective immunoglobulin G antibodies were unaffected by the Ile-186 substitution but poorly recognized HA with Lys-156. Furthermore, memory AFC responsiveness was localized to regional lymphoid tissue in the upper respiratory tract, where challenge HA was found. Thus, it is recommended that in the selection of vaccine candidates, virus populations with the egg-adapted HA Lys-156 substitution be eliminated and that, instead, egg-grown isolates which minimally contain Ile-186 be used as logical alternatives to MDCK cell-grown viruses.

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Selected References

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  1. Allan W., Tabi Z., Cleary A., Doherty P. C. Cellular events in the lymph node and lung of mice with influenza. Consequences of depleting CD4+ T cells. J Immunol. 1990 May 15;144(10):3980–3986. [PubMed] [Google Scholar]
  2. Bachmann M. F., Kündig T. M., Odermatt B., Hengartner H., Zinkernagel R. M. Free recirculation of memory B cells versus antigen-dependent differentiation to antibody-forming cells. J Immunol. 1994 Oct 15;153(8):3386–3397. [PubMed] [Google Scholar]
  3. Banchereau J., Bazan F., Blanchard D., Brière F., Galizzi J. P., van Kooten C., Liu Y. J., Rousset F., Saeland S. The CD40 antigen and its ligand. Annu Rev Immunol. 1994;12:881–922. doi: 10.1146/annurev.iy.12.040194.004313. [DOI] [PubMed] [Google Scholar]
  4. Barnett B. C., Burt D. S., Graham C. M., Warren A. P., Skehel J. J., Thomas D. B. I-Ad restricted T cell recognition of influenza hemagglutinin. Synthetic peptides identify multiple epitopes corresponding to antibody-binding regions of the HA1 subunit. J Immunol. 1989 Oct 15;143(8):2663–2669. [PubMed] [Google Scholar]
  5. Barnett B. C., Graham C. M., Burt D. S., Skehel J. J., Thomas D. B. The immune response of BALB/c mice to influenza hemagglutinin: commonality of the B cell and T cell repertoires and their relevance to antigenic drift. Eur J Immunol. 1989 Mar;19(3):515–521. doi: 10.1002/eji.1830190316. [DOI] [PubMed] [Google Scholar]
  6. Bean W. J., Jr, Sriram G., Webster R. G. Electrophoretic analysis of iodine-labeled influenza virus RNA segments. Anal Biochem. 1980 Feb;102(1):228–232. doi: 10.1016/0003-2697(80)90343-7. [DOI] [PubMed] [Google Scholar]
  7. Brumeanu T. D., Swiggard W. J., Steinman R. M., Bona C. A., Zaghouani H. Efficient loading of identical viral peptide onto class II molecules by antigenized immunoglobulin and influenza virus. J Exp Med. 1993 Nov 1;178(5):1795–1799. doi: 10.1084/jem.178.5.1795. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Burt D. S., Mills K. H., Skehel J. J., Thomas D. B. Diversity of the class II (I-Ak/I-Ek)-restricted T cell repertoire for influenza hemagglutinin and antigenic drift. Six nonoverlapping epitopes on the HA1 subunit are defined by synthetic peptides. J Exp Med. 1989 Aug 1;170(2):383–397. doi: 10.1084/jem.170.2.383. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Caton A. J., Brownlee G. G., Yewdell J. W., Gerhard W. The antigenic structure of the influenza virus A/PR/8/34 hemagglutinin (H1 subtype). Cell. 1982 Dec;31(2 Pt 1):417–427. doi: 10.1016/0092-8674(82)90135-0. [DOI] [PubMed] [Google Scholar]
  10. Caton A. J., Gerhard W. The diversity of the CD4+ T cell response in influenza. Semin Immunol. 1992 Apr;4(2):85–90. [PubMed] [Google Scholar]
  11. Caton A. J., Stark S. E., Kavaler J., Staudt L. M., Schwartz D., Gerhard W. Many variable region genes are utilized in the antibody response of BALB/c mice to the influenza virus A/PR/8/34 hemagglutinin. J Immunol. 1991 Sep 1;147(5):1675–1686. [PubMed] [Google Scholar]
  12. Clarke S., Rickert R., Wloch M. K., Staudt L., Gerhard W., Weigert M. The BALB/c secondary response to the Sb site of influenza virus hemagglutinin. Nonrandom silent mutation and unequal numbers of VH and Vk mutations. J Immunol. 1990 Oct 1;145(7):2286–2296. [PubMed] [Google Scholar]
  13. Cosgrove D., Gray D., Dierich A., Kaufman J., Lemeur M., Benoist C., Mathis D. Mice lacking MHC class II molecules. Cell. 1991 Sep 6;66(5):1051–1066. doi: 10.1016/0092-8674(91)90448-8. [DOI] [PubMed] [Google Scholar]
  14. Czerkinsky C. C., Nilsson L. A., Nygren H., Ouchterlony O., Tarkowski A. A solid-phase enzyme-linked immunospot (ELISPOT) assay for enumeration of specific antibody-secreting cells. J Immunol Methods. 1983 Dec 16;65(1-2):109–121. doi: 10.1016/0022-1759(83)90308-3. [DOI] [PubMed] [Google Scholar]
  15. Gerhard W., Haberman A. M., Scherle P. A., Taylor A. H., Palladino G., Caton A. J. Identification of eight determinants in the hemagglutinin molecule of influenza virus A/PR/8/34 (H1N1) which are recognized by class II-restricted T cells from BALB/c mice. J Virol. 1991 Jan;65(1):364–372. doi: 10.1128/jvi.65.1.364-372.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Germain R. N. MHC-dependent antigen processing and peptide presentation: providing ligands for T lymphocyte activation. Cell. 1994 Jan 28;76(2):287–299. doi: 10.1016/0092-8674(94)90336-0. [DOI] [PubMed] [Google Scholar]
  17. Graham M. B., Braciale V. L., Braciale T. J. Influenza virus-specific CD4+ T helper type 2 T lymphocytes do not promote recovery from experimental virus infection. J Exp Med. 1994 Oct 1;180(4):1273–1282. doi: 10.1084/jem.180.4.1273. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Gray D., Siepmann K., Wohlleben G. CD40 ligation in B cell activation, isotype switching and memory development. Semin Immunol. 1994 Oct;6(5):303–310. doi: 10.1006/smim.1994.1039. [DOI] [PubMed] [Google Scholar]
  19. Gray D. The dynamics of immunological memory. Semin Immunol. 1992 Feb;4(1):29–34. [PubMed] [Google Scholar]
  20. Gubareva L. V., Wood J. M., Meyer W. J., Katz J. M., Robertson J. S., Major D., Webster R. G. Codominant mixtures of viruses in reference strains of influenza virus due to host cell variation. Virology. 1994 Feb 15;199(1):89–97. doi: 10.1006/viro.1994.1100. [DOI] [PubMed] [Google Scholar]
  21. Gupta R. K., Relyveld E. H., Lindblad E. B., Bizzini B., Ben-Efraim S., Gupta C. K. Adjuvants--a balance between toxicity and adjuvanticity. Vaccine. 1993;11(3):293–306. doi: 10.1016/0264-410x(93)90190-9. [DOI] [PubMed] [Google Scholar]
  22. Hayakawa K., Ishii R., Yamasaki K., Kishimoto T., Hardy R. R. Isolation of high-affinity memory B cells: phycoerythrin as a probe for antigen-binding cells. Proc Natl Acad Sci U S A. 1987 Mar;84(5):1379–1383. doi: 10.1073/pnas.84.5.1379. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Hou S., Mo X. Y., Hyland L., Doherty P. C. Host response to Sendai virus in mice lacking class II major histocompatibility complex glycoproteins. J Virol. 1995 Mar;69(3):1429–1434. doi: 10.1128/jvi.69.3.1429-1434.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Hyland L., Hou S., Coleclough C., Takimoto T., Doherty P. C. Mice lacking CD8+ T cells develop greater numbers of IgA-producing cells in response to a respiratory virus infection. Virology. 1994 Oct;204(1):234–241. doi: 10.1006/viro.1994.1527. [DOI] [PubMed] [Google Scholar]
  25. Hyland L., Sangster M., Sealy R., Coleclough C. Respiratory virus infection of mice provokes a permanent humoral immune response. J Virol. 1994 Sep;68(9):6083–6086. doi: 10.1128/jvi.68.9.6083-6086.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Johansson B. E., Bucher D. J., Kilbourne E. D. Purified influenza virus hemagglutinin and neuraminidase are equivalent in stimulation of antibody response but induce contrasting types of immunity to infection. J Virol. 1989 Mar;63(3):1239–1246. doi: 10.1128/jvi.63.3.1239-1246.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Johansson B. E., Kilbourne E. D. Dissociation of influenza virus hemagglutinin and neuraminidase eliminates their intravirionic antigenic competition. J Virol. 1993 Oct;67(10):5721–5723. doi: 10.1128/jvi.67.10.5721-5723.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Johansson B. E., Kilbourne E. D. Influenza vaccine strain selection: equivalence of two antigenically distinct haemagglutinin variants of 1989 H3N2 influenza A virus in protection of mice. Vaccine. 1992;10(9):603–606. doi: 10.1016/0264-410x(92)90440-u. [DOI] [PubMed] [Google Scholar]
  29. Jones P. D., Ada G. L. Influenza-specific antibody-secreting cells and B cell memory in the murine lung after immunization with wild-type, cold-adapted variant and inactivated influenza viruses. Vaccine. 1987 Sep;5(3):244–248. doi: 10.1016/0264-410x(87)90109-5. [DOI] [PubMed] [Google Scholar]
  30. Katz J. M., Naeve C. W., Webster R. G. Host cell-mediated variation in H3N2 influenza viruses. Virology. 1987 Feb;156(2):386–395. doi: 10.1016/0042-6822(87)90418-1. [DOI] [PubMed] [Google Scholar]
  31. Katz J. M., Wang M., Webster R. G. Direct sequencing of the HA gene of influenza (H3N2) virus in original clinical samples reveals sequence identity with mammalian cell-grown virus. J Virol. 1990 Apr;64(4):1808–1811. doi: 10.1128/jvi.64.4.1808-1811.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Katz J. M., Webster R. G. Amino acid sequence identity between the HA1 of influenza A (H3N2) viruses grown in mammalian and primary chick kidney cells. J Gen Virol. 1992 May;73(Pt 5):1159–1165. doi: 10.1099/0022-1317-73-5-1159. [DOI] [PubMed] [Google Scholar]
  33. Katz J. M., Webster R. G. Efficacy of inactivated influenza A virus (H3N2) vaccines grown in mammalian cells or embryonated eggs. J Infect Dis. 1989 Aug;160(2):191–198. doi: 10.1093/infdis/160.2.191. [DOI] [PubMed] [Google Scholar]
  34. Kavaler J., Caton A. J., Staudt L. M., Schwartz D., Gerhard W. A set of closely related antibodies dominates the primary antibody response to the antigenic site CB of the A/PR/8/34 influenza virus hemagglutinin. J Immunol. 1990 Oct 1;145(7):2312–2321. [PubMed] [Google Scholar]
  35. Kawabe T., Naka T., Yoshida K., Tanaka T., Fujiwara H., Suematsu S., Yoshida N., Kishimoto T., Kikutani H. The immune responses in CD40-deficient mice: impaired immunoglobulin class switching and germinal center formation. Immunity. 1994 Jun;1(3):167–178. doi: 10.1016/1074-7613(94)90095-7. [DOI] [PubMed] [Google Scholar]
  36. Kilbourne E. D., Johansson B. E., Moran T., Wu S., Pokorny B. A., Xu X., Cox N. Influenza A virus haemagglutinin polymorphism: pleiotropic antigenic variants of A/Shanghai/11/87 (H3N2) virus selected as high yield reassortants. J Gen Virol. 1993 Jul;74(Pt 7):1311–1316. doi: 10.1099/0022-1317-74-7-1311. [DOI] [PubMed] [Google Scholar]
  37. Liang S., Mozdzanowska K., Palladino G., Gerhard W. Heterosubtypic immunity to influenza type A virus in mice. Effector mechanisms and their longevity. J Immunol. 1994 Feb 15;152(4):1653–1661. [PubMed] [Google Scholar]
  38. MacLennan I. C. Germinal centers. Annu Rev Immunol. 1994;12:117–139. doi: 10.1146/annurev.iy.12.040194.001001. [DOI] [PubMed] [Google Scholar]
  39. MacLennan I. C., Liu Y. J., Oldfield S., Zhang J., Lane P. J. The evolution of B-cell clones. Curr Top Microbiol Immunol. 1990;159:37–63. doi: 10.1007/978-3-642-75244-5_3. [DOI] [PubMed] [Google Scholar]
  40. McHeyzer-Williams M. G., McLean M. J., Lalor P. A., Nossal G. J. Antigen-driven B cell differentiation in vivo. J Exp Med. 1993 Jul 1;178(1):295–307. doi: 10.1084/jem.178.1.295. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Meyer W. J., Wood J. M., Major D., Robertson J. S., Webster R. G., Katz J. M. Influence of host cell-mediated variation on the international surveillance of influenza A (H3N2) viruses. Virology. 1993 Sep;196(1):130–137. doi: 10.1006/viro.1993.1461. [DOI] [PubMed] [Google Scholar]
  42. Newman R. W., Jennings R., Major D. L., Robertson J. S., Jenkins R., Potter C. W., Burnett I., Jewes L., Anders M., Jackson D. Immune response of human volunteers and animals to vaccination with egg-grown influenza A (H1N1) virus is influenced by three amino acid substitutions in the haemagglutinin molecule. Vaccine. 1993;11(4):400–406. doi: 10.1016/0264-410x(93)90279-7. [DOI] [PubMed] [Google Scholar]
  43. Ozaki S., Berzofsky J. A. Antibody conjugates mimic specific B cell presentation of antigen: relationship between T and B cell specificity. J Immunol. 1987 Jun 15;138(12):4133–4142. [PubMed] [Google Scholar]
  44. Paul W. E., Seder R. A. Lymphocyte responses and cytokines. Cell. 1994 Jan 28;76(2):241–251. doi: 10.1016/0092-8674(94)90332-8. [DOI] [PubMed] [Google Scholar]
  45. Robertson J. S., Bootman J. S., Newman R., Oxford J. S., Daniels R. S., Webster R. G., Schild G. C. Structural changes in the haemagglutinin which accompany egg adaptation of an influenza A(H1N1) virus. Virology. 1987 Sep;160(1):31–37. doi: 10.1016/0042-6822(87)90040-7. [DOI] [PubMed] [Google Scholar]
  46. Robertson J. S., Cook P., Nicolson C., Newman R., Wood J. M. Mixed populations in influenza virus vaccine strains. Vaccine. 1994 Nov;12(14):1317–1322. doi: 10.1016/s0264-410x(94)80058-8. [DOI] [PubMed] [Google Scholar]
  47. Robertson J. S., Naeve C. W., Webster R. G., Bootman J. S., Newman R., Schild G. C. Alterations in the hemagglutinin associated with adaptation of influenza B virus to growth in eggs. Virology. 1985 May;143(1):166–174. doi: 10.1016/0042-6822(85)90105-9. [DOI] [PubMed] [Google Scholar]
  48. Robertson J. S., Nicolson C., Newman R., Major D., Dunleavy U., Wood J. M. High growth reassortant influenza vaccine viruses: new approaches to their control. Biologicals. 1992 Sep;20(3):213–220. doi: 10.1016/s1045-1056(05)80040-5. [DOI] [PubMed] [Google Scholar]
  49. Rocha E., Cox N. J., Black R. A., Harmon M. W., Harrison C. J., Kendal A. P. Antigenic and genetic variation in influenza A (H1N1) virus isolates recovered from a persistently infected immunodeficient child. J Virol. 1991 May;65(5):2340–2350. doi: 10.1128/jvi.65.5.2340-2350.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  50. Rota P. A., Shaw M. W., Kendal A. P. Cross-protection against microvariants of influenza virus type B by vaccinia viruses expressing haemagglutinins from egg- or MDCK cell-derived subpopulations of influenza virus type B/England/222/82. J Gen Virol. 1989 Jun;70(Pt 6):1533–1537. doi: 10.1099/0022-1317-70-6-1533. [DOI] [PubMed] [Google Scholar]
  51. Ryan-Poirier K. A., Kawaoka Y. Distinct glycoprotein inhibitors of influenza A virus in different animal sera. J Virol. 1991 Jan;65(1):389–395. doi: 10.1128/jvi.65.1.389-395.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  52. Sangster M., Hyland L., Sealy R., Coleclough C. Distinctive kinetics of the antibody-forming cell response to Sendai virus infection of mice in different anatomical compartments. Virology. 1995 Feb 20;207(1):287–291. doi: 10.1006/viro.1995.1079. [DOI] [PubMed] [Google Scholar]
  53. Scherle P. A., Gerhard W. Functional analysis of influenza-specific helper T cell clones in vivo. T cells specific for internal viral proteins provide cognate help for B cell responses to hemagglutinin. J Exp Med. 1986 Oct 1;164(4):1114–1128. doi: 10.1084/jem.164.4.1114. [DOI] [PMC free article] [PubMed] [Google Scholar]
  54. Sedgwick J. D., Holt P. G. A solid-phase immunoenzymatic technique for the enumeration of specific antibody-secreting cells. J Immunol Methods. 1983 Feb 25;57(1-3):301–309. doi: 10.1016/0022-1759(83)90091-1. [DOI] [PubMed] [Google Scholar]
  55. Snapper C. M., Mond J. J. Towards a comprehensive view of immunoglobulin class switching. Immunol Today. 1993 Jan;14(1):15–17. doi: 10.1016/0167-5699(93)90318-F. [DOI] [PubMed] [Google Scholar]
  56. Sprent J. T and B memory cells. Cell. 1994 Jan 28;76(2):315–322. doi: 10.1016/0092-8674(94)90338-7. [DOI] [PubMed] [Google Scholar]
  57. Stark S. E., Caton A. J. Antibodies that are specific for a single amino acid interchange in a protein epitope use structurally distinct variable regions. J Exp Med. 1991 Sep 1;174(3):613–624. doi: 10.1084/jem.174.3.613. [DOI] [PMC free article] [PubMed] [Google Scholar]
  58. Staudt L. M., Gerhard W. Generation of antibody diversity in the immune response of BALB/c mice to influenza virus hemagglutinin. I. Significant variation in repertoire expression between individual mice. J Exp Med. 1983 Feb 1;157(2):687–704. doi: 10.1084/jem.157.2.687. [DOI] [PMC free article] [PubMed] [Google Scholar]
  59. Stern L. J., Brown J. H., Jardetzky T. S., Gorga J. C., Urban R. G., Strominger J. L., Wiley D. C. Crystal structure of the human class II MHC protein HLA-DR1 complexed with an influenza virus peptide. Nature. 1994 Mar 17;368(6468):215–221. doi: 10.1038/368215a0. [DOI] [PubMed] [Google Scholar]
  60. Temoltzin-Palacios F., Thomas D. B. Modulation of immunodominant sites in influenza hemagglutinin compromise antigenic variation and select receptor-binding variant viruses. J Exp Med. 1994 May 1;179(5):1719–1724. doi: 10.1084/jem.179.5.1719. [DOI] [PMC free article] [PubMed] [Google Scholar]
  61. Tew J. G., DiLosa R. M., Burton G. F., Kosco M. H., Kupp L. I., Masuda A., Szakal A. K. Germinal centers and antibody production in bone marrow. Immunol Rev. 1992 Apr;126:99–112. doi: 10.1111/j.1600-065x.1992.tb00633.x. [DOI] [PubMed] [Google Scholar]
  62. Walker R. I. New strategies for using mucosal vaccination to achieve more effective immunization. Vaccine. 1994 Apr;12(5):387–400. doi: 10.1016/0264-410x(94)90112-0. [DOI] [PubMed] [Google Scholar]
  63. Wang Y., Smith J. A., Gefter M. L., Perkins D. L. Immunodominance: intermolecular competition between MHC class II molecules by covalently linked T cell epitopes. J Immunol. 1992 May 15;148(10):3034–3041. [PubMed] [Google Scholar]
  64. Watts C., Lanzavecchia A. Suppressive effect of antibody on processing of T cell epitopes. J Exp Med. 1993 Oct 1;178(4):1459–1463. doi: 10.1084/jem.178.4.1459. [DOI] [PMC free article] [PubMed] [Google Scholar]
  65. Wiley D. C., Wilson I. A., Skehel J. J. Structural identification of the antibody-binding sites of Hong Kong influenza haemagglutinin and their involvement in antigenic variation. Nature. 1981 Jan 29;289(5796):373–378. doi: 10.1038/289373a0. [DOI] [PubMed] [Google Scholar]
  66. Wilson I. A., Cox N. J. Structural basis of immune recognition of influenza virus hemagglutinin. Annu Rev Immunol. 1990;8:737–771. doi: 10.1146/annurev.iy.08.040190.003513. [DOI] [PubMed] [Google Scholar]
  67. Wilson I. A., Skehel J. J., Wiley D. C. Structure of the haemagglutinin membrane glycoprotein of influenza virus at 3 A resolution. Nature. 1981 Jan 29;289(5796):366–373. doi: 10.1038/289366a0. [DOI] [PubMed] [Google Scholar]
  68. Wood J. M., Oxford J. S., Dunleavy U., Newman R. W., Major D., Robertson J. S. Influenza A (H1N1) vaccine efficacy in animal models is influenced by two amino acid substitutions in the hemagglutinin molecule. Virology. 1989 Jul;171(1):214–221. doi: 10.1016/0042-6822(89)90528-x. [DOI] [PubMed] [Google Scholar]
  69. al-Shakhshir R., Regnier F., White J. L., Hem S. L. Effect of protein adsorption on the surface charge characteristics of aluminium-containing adjuvants. Vaccine. 1994 Apr;12(5):472–474. doi: 10.1016/0264-410x(94)90127-9. [DOI] [PubMed] [Google Scholar]
  70. van den Eertwegh A. J., Laman J. D., Noelle R. J., Boersma W. J., Claassen E. In vivo T-B cell interactions and cytokine-production in the spleen. Semin Immunol. 1994 Oct;6(5):327–336. doi: 10.1006/smim.1994.1041. [DOI] [PubMed] [Google Scholar]

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