Development of a prophylactic vaccine against Epstein-Barr virus (EBV)
Experimental plan. We have produced a DNA construct that encodes for two identical copies of the EBV gp350 protein separated by a spacer. 3 to the second gp350 are sequences encoding for 2 tetanus toxoid (TT) peptides that contain dominant epitopes for recruiting CD4+ T cell help. A leucine zipper dimerization domain has been placed 3 to the TT sequences in order to produce a final protein that will be tetrameric for gp350. As explained below, multimerization of gp350 will potentially effect B cell receptor-CD21 interactions in cis, for synergistic B cell signaling, and enhance binding of antigen to follicular dendritic cells (FDC) that also express CD21, for enhanced retention of antigen within germinal centers. Collectively this design should result in a more vigorous IgG anti-gp350 antibody response in vivo than that resulting from immunization with monomeric gp350 alone. CHO cells have been transfected with this gp350 DNA construct and protein has been expressed for testing in mice and monkeys.
EBV gp350 is a potential target protein for a prophylactic EBV vaccine. EBV is a major, global source of morbidity and mortality, responsible for such pathologic entities as Burkitt lymphoma, nasopharyngeal carcinoma, infectious mononucleosis, a subset of Hodgkin's disease, and the lymphoproliferative syndrome in immunosuppressed patients (1-3). In the developing world, EBV seroconversion typically occurs in infancy, whereas in developed countries it is more likely contracted in adolescence. Infectious mononucleosis typically occurs only in this latter group (3). The major human reservoir for latent EBV and EBV transmission is the resting memory B lymphocyte (4). EBV is dependent upon the gp350-CD21 binding event for viral entry into the B cell (5, 6), an event that is critical for infectivity and B cell neoplastic transformation (2). Sera from patients with active EBV infection contain antibody that prevent EBV entry into B cells (neutralizing antibody). Adsorption of these sera with gp350, eliminates most of this neutralizing activity (7), indicating that gp350 serves as the major EBV antigen to which a protective humoral immune response is directed.
Co-crosslinking of antigen receptor and complement receptor type 2 (CD21) is synergistic for B cell activation. CD21 binds the C3 degradation product C3d. Under physiologic conditions C3d may be generated, and bind, directly to the surface of bacteria or viruses via the alternative activation pathway. Additionally, immune complexes can fix C3d via the classical pathway of activation (8, 9). Mice genetically deficient in CD21 exhibit defective T cell-dependent antibody responses both in vitro and in vivo (10, 11). Furthermore, in vivo antibody responses are inhibited by soluble forms of CD21 or anti-CD21 antibody (12). The adjuvanting properties of C3d bound to CD21 are due both to its ability to lower the threshold of B cell activation stimulated by antigen, as well as its binding to follicular dendritic cells which trap and present antigen within germinal centers (13-15). Indeed, antigens bound to C3d induce B cell activation at up to 1000-10,000-fold lower concentrations of antigen as compared to antigen only (16-20). Cross-linking of the B cell CD21 by anti-CD21 antibodies has also been shown to induce B cell proliferation and differentiation in the presence of T cell factors, even in the absence of an mIg signaling event (13, 21, 22). In this regard, mice immunized with protein or polysaccharide antigens covalently linked to at least 2 molecules of C3d elicit strikingly higher antibody responses to these antigens, relative to immunization with antigen alone (19, 20, 23).
Epstein-Barr virus (EBV)-derived gp350 binds to human CD21 and can co-stimulate B cell activation. EBV expresses the gp350 envelope glycoprotein which binds to CD21 (24, 25). Covalent linkage, but not mixing, of anti-human IgD to gp350 was shown to stimulate higher levels of human B cell proliferation at 10-fold lower concentrations of anti-human IgD, relative to anti-human IgD alone (26). This effect was specifically blocked with anti-CD21 antibody (26). Covalent linkage of the FG protein of respiratory syncytial virus to anti-IgD, to serve as a negative control, had no affect on B cell proliferation relative to anti-IgD alone. Additionally, anti-IgD-gp350 stimulated a more sustained calcium flux as compared to unconjugated anti-IgD (26). Thus, we predict that multimeric gp350 will co-crosslink CD21 with the B cell receptor on gp350-specific B cells for synergistic B cell signaling.
Pilot studies in non-human primates and humans suggest the feasibility of using gp350 for conferring protection against EBV-mediated diseases. A number of studies have demonstrated that immunization of non-human primates with a subunit gp350 vaccine in adjuvant protects against experimental EBV-induced lymphoma or EBV replication. Thus, purified native gp350, injected into cottontop marmosets (CTM), in association with liposomes, ISCOM's, or muramyl dipeptide, protected against EBV-induced lymphoma (27, 28). Recombinant gp350 in alum or muramyl dipeptide was similarly protective (29, 30). Common marmosets also showed decreased viral replication after EBV challenge following immunization with recombinant gp350 in alum (31). Non-human primate studies using gp350 expressed by adenoviral or vaccinia vectors have similarly shown protection against experimental EBV-induced lymphoma or EBV replication in CTM or common marmosets (32-34).
An important qualification to the above studies, is that the development of neutralizing antibody to gp350 in tamarins did not always correlate with protection. Thus, some adenovirus-gp350 or vaccinia-gp350 vaccinated animals were protected even without neutralizing antibody to gp350 (33, 34). Additionally, some tamarins vaccinated with gp350 purified from infected cells, produced neutralizing antibody to gp350, but were not protected (35). These data suggest that CTLs to gp350 may also be important in primate models of lymphoma.
A pilot study in humans has also suggested a potential role for gp350 vaccination in host protection against EBV. In a study by Gu et al (36) a single dose of gp350/220 expressed by vaccinia virus (VV) was give by scarification to 1-3 year olds who were EBV-seronegative, and VV-seronegative. These children developed neutralizing antibodies to EBV (1:40-1:160). Whereas 10/10 unvaccinated controls became infected at 16 months of follow-up, only 3/9 vaccinated children became infected at this time. More recently a small phase 2 clinical trial suggested the ability of gp350 vaccination to reduce the incidence of infectious mononucleosis, though not asymptomatic EBV infection, of previously seronegative young adults (37, 38).
- 1. Cohen, J. I. 1999. The biology of Epstein-Barr virus: lessons learned from the virus and the host. Curr Opin Immunol 11:365-370.
- 2. Thorley-Lawson, D. A. 2005. EBV the prototypical human tumor virus--just how bad is it? J Allergy Clin Immunol 116:251-261; quiz 262.
- 3. Vetsika, E. K., and M. Callan. 2004. Infectious mononucleosis and Epstein-Barr virus. Expert Rev Mol Med 6:1-16.
- 4. Babcock, G. J., L. L. Decker, M. Volk, and D. A. Thorley-Lawson. 1998. EBV persistence in memory B cells in vivo. Immunity 9:395-404.
- 5. Tanner, J., J. Weis, D. Fearon, Y. Whang, and E. Kieff. 1987. Epstein-Barr virus gp350/220 binding to the B lymphocyte C3d receptor mediates adsorption, capping, and endocytosis. Cell 50:203-213.
- 6. Tanner, J., Y. Whang, J. Sample, A. Sears, and E. Kieff. 1988. Soluble gp350/220 and deletion mutant glycoproteins block Epstein-Barr virus adsorption to lymphocytes. J Virol 62:4452-4464.
- 7. Thorley-Lawson, D. A., and C. A. Poodry. 1982. Identification and isolation of the main component (gp350-gp220) of Epstein-Barr virus responsible for generating neutralizing antibodies in vivo. J Virol 43:730-736.
- 8. Tolnay, M., and G. C. Tsokos. 1998. Complement receptor 2 in the regulation of the immune response. Clin Immunol Immunopathol 88:123-132.
- 9. Muller-Eberhard, H. J. 1988. Molecular organization and function of the complement system. Annu Rev Biochem 57:321-347.
- 10. Croix, D. A., J. M. Ahearn, A. M. Rosengard, S. Han, G. Kelsoe, M. Ma, and M. C. Carroll. 1996. Antibody response to a T-dependent antigen requires B cell expression of complement receptors. J. Exp. Med. 183:1857-1864.
- 11. Molina, H., V. M. Holers, B. Li, Y. Fung, S. Mariathasan, J. Goellner, J. Strauss-Schoenberger, R. W. Karr, and D. D. Chaplin. 1996. Markedly impaired humoral immune response in mice deficient in complement receptors 1 and 2. Proc Natl Acad Sci U S A 93:3357-3361.
- 12. Hebell, T., J. M. Ahearn, and D. T. Fearon. 1991. Suppression of the immune response by a soluble
- 13. Fearon, D. T., and R. H. Carter. 1995. The CD19/CR2/TAPA-1 complex of B lymphocytes: linking natural to acquired immunity. Annu Rev Immunol 13:127-149.
- 14. Carroll, M. C., and M. B. Fischer. 1997. Complement and the immune response. Curr Opin Immunol 9:64-69.
- 15. Bohnsack, J. F., and N. R. Cooper. 1988. CR2 ligands modulate human B cell activation. J Immunol 141:2569-2576.
- 16. Carter, R. H., and D. T. Fearon. 1989. Polymeric C3dg primes human B lymphocytes for proliferation induced by anti-IgM. J Immunol 143:1755-1760.
- 17. Carter, R. H., M. O. Spycher, Y. C. Ng, R. Hoffman, and D. T. Fearon. 1988. Synergistic interaction between complement receptor type 2 and membrane IgM on B lymphocytes. J Immunol 141:457-463.
- 18. Mongini, P. K. A., M. A. Vilensky, P. F. Highet, and J. K. Inman. 1997. The affinity threshold for human B cell activation via the Antigen receptor complex is reduced upon co-ligation of the antigen receptor with CD21 (CR2). J. Immunol. 159:3782-3791.
- 19. Test, S. T., J. Mitsuyoshi, C. C. Connolly, and A. H. Lucas. 2001. Increased immunogenicity and induction of class switching by conjugation of complement C3d to pneumococcal serotype 14 capsular polysaccharide. Infect Immun 69:3031-3040.
- 20. Dempsey, P. W., M. E. Allison, S. Akkaraju, C. C. Goodnow, and D. T. Fearon. 1996. C3d of complement as a molecular adjuvant: bridging innate and acquired immunity. Science 271:348-350..
- 21. Frade, R., M. C. Crevon, M. Barel, A. Vazquez, L. Krikorian, C. Charriaut, and P. Galanaud. 1985. Enhancement of human B cell proliferation by an antibody to the C3d receptor, the gp 140 molecule. Eur J Immunol 15:73-76.
- 22. Wilson, B. S., J. L. Platt, and N. E. Kay. 1985. Monoclonal antibodies to the 140,000 mol wt glycoprotein of B lymphocyte membranes (CR2 receptor) initiates proliferation of B cells in vitro. Blood 66:824-829.
- 23. Griffioen, A. W. G., 1997 #1947}, G. T. Rijkers, P. Janssens-Korpela, and B. J. Zegers. 1991. Pneumococcal polysaccharides complexed with C3d bind to human B lymphocytes via complement receptor type 2. Infect Immun 59:1839-1845.
- 24. Fingeroth, J. D., J. J. Weis, T. F. Tedder, J. L. Strominger, P. A. Biro, and D. T. Fearon. 1984. Epstein-Barr virus receptor of human B lymphocytes is the C3d receptor CR2. Proc Natl Acad Sci U S A 81:4510-4514.
- 25. Nemerow, G. R., C. Mold, V. K. Schwend, V. Tollefson, and N. R. Cooper. 1987. Identification of gp350 as the viral glycoprotein mediating attachment of Epstein-Barr virus (EBV) to the EBV/C3d receptor of B cells: sequence homology of gp350 and C3 complement fragment C3d. J Virol 61:1416-1420.
- 26. Goeckeritz, B. E., A. Lees, Q. Vos, G. C. Tsokos, K. Kuhlbusch, and J. J. Mond. 2000. Enhanced and sustained activation of human B cells by anti- immunoglobulin conjugated to the EBV glycoprotein gp350. Eur J Immunol 30:969-973.
- 27. Morgan, A. J., M. A. Epstein, and J. R. North. 1984. Comparative immunogenicity studies on Epstein-Barr virus membrane antigen (MA) gp340 with novel adjuvants in mice, rabbits, and cotton-top tamarins. J Med Virol 13:281-292.
- 28. Morgan, A. J., A. C. Allison, S. Finerty, F. T. Scullion, N. E. Byars, and M. A. Epstein. 1989. Validation of a first-generation Epstein-Barr virus vaccine preparation suitable for human use. J Med Virol 29:74-78.
- 29. Finerty, S., J. Tarlton, M. Mackett, M. Conway, J. R. Arrand, P. E. Watkins, and A. J. Morgan. 1992. Protective immunization against Epstein-Barr virus-induced disease in cottontop tamarins using the virus envelope glycoprotein gp340 produced from a bovine papillomavirus expression vector. J Gen Virol 73 ( Pt 2):449-453.
- 30. Finerty, S., M. Mackett, J. R. Arrand, P. E. Watkins, J. Tarlton, and A. J. Morgan. 1994. Immunization of cottontop tamarins and rabbits with a candidate vaccine against the Epstein-Barr virus based on the major viral envelope glycoprotein gp340 and alum. Vaccine 12:1180-1184.
- 31. Cox, C., B. A. Naylor, M. Mackett, J. R. Arrand, B. E. Griffin, and N. Wedderburn. 1998. Immunization of common marmosets with Epstein-Barr virus (EBV) envelope glycoprotein gp340: effect on viral shedding following EBV challenge. J Med Virol 55:255-261.
- 32. Mackett, M., C. Cox, S. D. Pepper, J. F. Lees, B. A. Naylor, N. Wedderburn, and J. R. Arrand. 1996. Immunisation of common marmosets with vaccinia virus expressing Epstein-Barr virus (EBV) gp340 and challenge with EBV. J Med Virol 50:263-271.
- 33. Ragot, T., S. Finerty, P. E. Watkins, M. Perricaudet, and A. J. Morgan. 1993. Replication-defective recombinant adenovirus expressing the Epstein-Barr virus (EBV) envelope glycoprotein gp340/220 induces protective immunity against EBV-induced lymphomas in the cottontop tamarin. J Gen Virol 74 ( Pt 3):501-507.
- 34. Morgan, A. J., M. Mackett, S. Finerty, J. R. Arrand, F. T. Scullion, and M. A. Epstein. 1988. Recombinant vaccinia virus expressing Epstein-Barr virus glycoprotein gp340 protects cottontop tamarins against EB virus-induced malignant lymphomas. J Med Virol 25:189-195.
- 35. Epstein, M. A., B. J. Randle, S. Finerty, and J. K. Kirkwood. 1986. Not all potently neutralizing, vaccine-induced antibodies to Epstein-Barr virus ensure protection of susceptible experimental animals. Clin Exp Immunol 63:485-490.
- 36. Gu, S. Y., T. M. Huang, L. Ruan, Y. H. Miao, H. Lu, C. M. Chu, M. Motz, and H. Wolf. 1995. First EBV vaccine trial in humans using recombinant vaccinia virus expressing the major membrane antigen. Dev Biol Stand 84:171-177.
- 37. Sokal, E. M., K. Hoppenbrouwers, C. Vandermeulen, M. Moutschen, P. Leonard, A. Moreels, M. Haumont, A. Bollen, F. Smets, and M. Denis. 2007. Recombinant gp350 vaccine for infectious mononucleosis: a phase 2, randomized, double-blind, placebo-controlled trial to evaluate the safety, immunogenicity, and efficacy of an Epstein-Barr virus vaccine in healthy young adults. J Infect Dis 196:1749-1753.
- 38. Moutschen, M., P. Leonard, E. M. Sokal, F. Smets, M. Haumont, P. Mazzu, A. Bollen, F. Denamur, P. Peeters, G. Dubin, and M. Denis. 2007. Phase I/II studies to evaluate safety and immunogenicity of a recombinant gp350 Epstein-Barr virus vaccine in healthy adults. Vaccine 25:4697-4705.